Immunotherapeutic potential of modified lipooligosaccharides/lipid A

ABSTRACT

Embodiments of the disclosure provide for unique lipooligosaccharide/lipid A-based mimetics for use as adjuvants. Methods of generating lipooligosaccharide/lipid A-based mimetics are provided that utilize recombinantly engineered bacteria to produce the mimetics, including, for example, addition of one or more particular enzymes such as acyltransferases, deacylases, phosphatases, or glycosyltransferases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Non-Provisional patentapplication Ser. No. 14/772,282 filed Sep. 2, 2015, now U.S. patent Ser.No. 10/358,667, which is a national phase application under 35 U.S.C. §371 that claims priority to International Application No.PCT/US2014/022121 filed Mar. 7, 2014 which claims the benefit ofpriority to U.S. Provisional Patent Application No. 61/773,928, filedMar. 7, 2013, all of which application is are incorporated by referenceherein in its their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant NumberAI101685 awarded by The National Institutes of Health. The governmenthas certain rights in the invention.

TECHNICAL FIELD

Embodiments of the disclosure generally relate at least to the fields ofimmunology, cell biology, molecular biology, and medicine. The presentdisclosure generally relates to compositions, methods of screening, andmethods of use for immunotherapeutic molecules and adjuvants forimmunogenic formulations. More specifically, the present inventionrelates to compositions and methods of screening for immunogenicformulations using bacterial enzymatic combinatorial chemistry.

BACKGROUND OF THE INVENTION

To date, vaccine adjuvants such as complete Freund's adjuvant derivedfrom the cell wall of mycobacteria in a water-in-oil emulsion oraluminum salts have been developed using an empirical trial-and-errorapproach. This approach has identified compounds that compensate forpoor immune responsiveness to an antigen, increase vaccine stability,and reduce the dose of an antigen required for protection, though theexact immunological mechanism for their function in the host is stillunder debate. However, identifying or developing new,rationally-designed adjuvant(s) that stimulate components of the hostinnate and/or adaptive immune systems, based on known correlates ofimmune protection are now possible with efficacy and safety being theprimary goals. The significance of identifying novel adjuvants is highlyimportant as most of the current trends in vaccine development are basedon poorly immunogenic, highly purified antigens, which will require anappropriate adjuvant to induce an effective protective immunity.

An area of great interest is in the development of morerationally-designed adjuvants based on molecules that stimulate the hostinnate immune system, specifically pattern-recognition receptors,including toll-like receptors (TLR). The TLR family plays a criticalrole in early innate immunity by acting as sensors in response toinvading pathogens and are expressed in tissues involved in immunefunction, e.g. peripheral blood leukocytes and spleen or those exposedto the external environment like the gastrointestinal tract and lung. Todate, ten human and twelve murine TLRs have been identified with mostlocalized to the cell plasma membrane with the exception of TLR3, TLR7,TLR8, and TLR9 being localized intracellularly. Each receptor recognizeshighly conserved structural motifs expressed by microbial pathogens(PAMPs) that are different from host ligands. Briefly, TLR2 is essentialfor the recognition of bacterial lipoproteins, lipoarabinomannans andlipoteichoic acids from Gram-positive organism; TLR3 recognizes viraldsRNA; TLR5 detects flagellin, the major protein subunit of flagella;TLR9 recognizes hypo-methylated CpG DNA motifs; TLR7 and TLR8 recognizesmall synthetically derived viral RNAs; TLR4 is activated by LPS throughits bioactive component lipid A or endotoxin, in conjunction with theaccessory molecules, MD-2 and CD14 that form a complex with TLR4.Finally, TLR signaling specificities are extended by their ability toheterodimerize (they mostly homodimerize, but TLR2 heterodimerizes) withone another. Stimulation of TLRs by PAMPs initiates signaling cascadesthat involve a number of proteins, such as MyD88, TRIF and IRAK (Kawaiand Akira, 2011; Pasare and Medzhitov, 2005). These signaling cascadeslead to the activation of transcription factors, such as AP-1, NF-κB andIRFs inducing the secretion of pro-inflammatory cytokines, chemokines,and effector cytokines that direct or modify the host immune response.Among TLRs, only TLR3 and TLR4 stimulate the production of type I IFNsvia TRW and the induction of a robust IL-12p70 response that stronglyenhances cellular-mediated and humoral immune responses.

Currently, a number of TLR mimetics are being used as stand-aloneimmunotherapeutic adjuvants or in combination with TLR signalingmolecules. Examples include natural and synthetic-based lipid Amimetics, monophosphoryl lipid A (MPL) and aminoalkyl glucosaminidephosphates (AGPs). MPL is a chemically modified form of lipid A, derivedfrom Salmonella minnesota R595 lipopolysaccharide. These chemicalmodifications result in the generation of a 4′-monophosphoryl,3-O-deacylated lipid A structure that also displays differences in theoverall number and location of individual fatty acids attached to theglucosamine sugar backbone of lipid A. As MPL is chemically derived,lot-to-lot differences in the microheterogeneity of the acyl groups makeperforming structure-activity relationship studies problematic. Incontrast, the AGP classes of lipids are monosaccharide lipid A mimeticsbased on the biologically active hexa-acylated component present in MPL,chemically synthesized with modifications in the acyl chain length andlocation in uniform positions. Both molecules display low-toxicity, ascompared to LPS (approximately 0.1% as toxic as LPS for MPL) and arepotent immunostimulators of the host innate and adaptive immune system.Assessment of the adjuvant characteristics of MPL has shown it to be aneffective adjuvant for the induction of both humoral and cell-mediatedimmunity in which MPL can induce both Th₁- and Th₂-type immune responsesin the systemic and mucosal compartments of the immune system. MPL iscurrently a component in many of GalaxoSmithKline's (GSK) proprietaryand novel adjuvant systems used in multiple GSK Bio vaccines. However,due to the increased heterogeneity from the chemical hydrolysis of lipidA for the production of MPL and the limitations and labor intense natureof synthesizing AGPs that more closely mimic the structure of naturallyoccurring lipid A structures, alternative technologies are in need.

BRIEF SUMMARY OF THE INVENTION

Protection from infectious agents known to be major causes of deathworldwide, such as influenza, tuberculosis, and malaria, as well aspotential release of bioweaponized agents that cause plague, tularemia,and melioidosis, require vaccines that generate humoral and T-cellresponses. Effective component vaccines require the addition ofadjuvants to increase their immunogenic capacities. Until recently, alumsalts, which require repeated applications and tend to be skewed towardsT helper TH 2-based immunity (humoral) rather than TH 1, (cellular) werethe only adjuvants approved for use in human vaccines. Recently, thelipid A mimetic (monophosphoryl lipid A, MPL) adjuvant has been combinedwith alum (AS04) in two FDA-approved vaccines (Cervarix (Human PapillomaVirus), and Fendrix (Hepatitis B Virus)). Additionally, synthetic lipidA mimetics aminoalkyl glucosaminide phosphates (AGPs) that also signalthrough Toll-Like Receptor 4 (TLR4) are being studied as both adjuvantsor stand-alone immunogenic compounds. Thus, TLR4 agonists show greatpromise for use as adjuvants in component vaccines. However, theapproved TLR4 agonist, MPL, has distinct deficiencies both in potencyand structural consistency, and AGPs are labor intensive and costly tosynthesize.

The present disclosure is directed to compositions and methods relatedto immunological compositions and synthesis thereof, includingcompositions and methods related to adjuvants and their methods ofmaking them. Kits including the adjuvants are encompassed in thedisclosure. In particular embodiments, there are methods andcompositions related to modified lipooligosaccharides/Lipid A moleculesas adjuvants; the compositions may be considered to belipooligosaccharide/lipid A-based immunomodulators orlipooligosaccharide/lipid A-based immunopotentiators.

Provided herein is a novel approach of using Bacterial EnzymaticCombinatorial Chemistry (BECC) to make rationally-designed lipid Astructures by modifying the lipid A structure of a lipopolysaccharide(LPS) or lipooligosaccharide (LOS) from a Gram negative bacteria (suchas an attenuated (BSL-2 approved) Yersinia pestis (Yp) strain). Ingeneral aspects, this approach uses the lipid A structure present inLPS/LOS synthesized in bacteria as a lead molecule or structure to bemodified by heterologous in trans expression of lipid A biosynthesisenzymes. These enzymes are obtained from a wide variety of bacterialbackgrounds with specificities for the removal or addition of fatty acidchain, phosphates moieties, and carbohydrates to the lipid A backbone.In particular aspects, one approach uses the non-stimulatory,hypoacylated, and bisphosphorylated lipid A structure present in LOSsynthesized by a Yp strain. As such, methods of the disclosure allow forthe safe, cost effective, and efficient design of molecules withimmunostimulatory use. One can test the immunotherapeutic use of thesenew molecules in vitro and in vivo to identify novel moleculesrepresenting adjuvants and/or immunomodulating reagents. One can alsoinclude well-characterized immunostimulants, such as MPL and known LPSstructures, as comparisons to the molecules synthesized by BECC. Theprotective innate/adaptive immune responses by this novel approach ofcreating new adjuvants has important implications at least in the fieldsof antigen recognition, formulation, and vaccine design.

One embodiment of the disclosure comprises a variety of modifiedlipooligosaccharides comprising core regions of LPS and lipid Amolecules, wherein the structures have adjuvant-like properties; in atleast some regards, the compositions are lipooligosaccharide/lipidA-based agonists. In specific embodiments, the structures areTLR4-signaling molecules. Certain properties for thelipooligosaccharides/Lipid A molecules that are useful include having adetectable proinflammatory response but low adverse reaction for anindividual. Particular embodiments allow establishment of a novelmechanism for adjuvant/vaccine design leading to well-defined,outcome-specific adjuvants that can elicit cellular immunity (T_(H)1 andT_(H)17-type immune responses) in addition to robust antibody productionin the systemic and mucosal compartments of the host. In specificembodiments, when the compositions are administered with an antigen,antigen-specific adaptive responses, TH1 and/or TH2 and/or antibodiesare produced.

In embodiments of the disclosure, rationally-designed adjuvants based onmolecules that stimulate the host innate immune system are used alone orin other immunogenic compositions to combat disease. Provided herein arechemically defined lipooligosaccharide/lipid A-based adjuvants that aresafe, easy to produce, and effective in conferring protection against awide variety of pathogenic bacteria and viruses, for example. Withmethods of the invention, one can engineer a wide range of adjuvantmolecules for use either as stand-alone immunotherapeutic molecules oradjuvants in immunogenic compositions, including vaccine formulations,for example.

Provided herein are oligosaccharides with modified lipid A structuresthat are immunogenic. Also provided are methods of screening forimmunotherapeutic molecules and adjuvants for formulations ofimmunogenic compositions, including vaccine compositions. Further, thepresent disclosure provides methods for using the identified compoundsfor immunotherapy as immunotherapeutic molecules and adjuvants—forvaccine formulations. In one embodiment of the disclosure, methods ofsynthesizing structures are described using biosynthesis pathwayspresent in Gram-negative bacteria based on the presence or absence ofspecific phosphate, acyl, and carbohydrate groups, as an example ofmodifications. Particular aspects of the methods involve the de novosynthesis of lipid A-like compositions in a Gram-negative bacteria, asopposed to modifications of a known or existing molecule.

In one embodiment of the disclosure, there are methods of screening forimmunotherapeutic molecules and adjuvants for vaccine formulations.

Embodiments of the disclosure exploit the lipid A biosynthetic pathwayof a Gram negative bacteria for the synthesis of novel TLR4-basedimmunostimulators. In certain aspects, the disclosure provides anunderstanding of the molecular basis by which Gram-negative bacteriamodify the lipid A component of lipopolysaccharide (LPS) and how thesealterations affect or circumvent normal host innate immune systemresponses. These modifications can promote resistance to host innateimmune killing mechanisms by antimicrobial compounds and alterrecognition by TLR4.

Particular aspects include modifications of LPS, the major component ofthe Gram-negative bacterial envelope, from P. aeruginosa, Francisellasubspecies, Bordetella subspecies, S. typhimurium, Acinetobacterbaumannii, Burkholderia, and Yersiniae subspecies, and in specificaspects the lipid A component of LPS is altered in one or more of thesebacteria using methods of the disclosure.

Embodiments of the disclosure include administering a therapeuticallyeffective amount of at least one modified lipooligosaccharide/lipid Acomposition to an individual in need thereof. The administration may beby any route, although in particular embodiments a variety ofadministrative routes (intramuscular, intravenous, intranasal, aerosol,subcutaneous, intraperitoneal, intradermal, for example) are employed.

The structure and/or function of lipooligosaccharide/lipid A-basedmimetics of the disclosure may be modified as compared to a hostbacteria in which the composition is generated or as compared to areference molecule, such as MPL. In specific aspects, candidate adjuvantmolecules are tested in vivo, including, for example, via an intranasalroute and, optionally, a subsequent intramuscular route. In certaincases, there is use of tissue culture cell lines (human and murine TLR4)as well as primary dendritic cells (human and murine-derived) toevaluate toxicity and proinflammatory responses to the BECC-synthesizedmolecules.

The modifications in the lipooligosaccharide/lipid A-based mimetics maybe of any kind, including modifications to the fatty acid content and/ornumber, the number of phosphates and/or modification thereof, and thenumber or type of sugar. In certain embodiments, the construction of newBECC-synthesized adjuvant molecules concern altering the terminalphosphates on the glucosamine backbone of lipid A that is useful foreliciting innate immune responses.

In specific aspects, the bacteria is an Archaebacteria. In specificembodiments, the bacteria is an extremophile, including an Acidophile;Alkaliphile; Anaerobe; Cryptoendolith; Halophile; Hyperthermophile;Hypolith; Lithoautotroph; Metallotolerant; Oligotroph; Osmophile;Piezophile; Polyextremophile; Psychrophile/Cryophile; Radioresistant;Thermoacidophile; or Xerophile, for example.

In particular aspects, the bacteria in which thelipooligosaccharide/lipid A-based mimetics are generated is an avirulentY. pestis strain, such as one that has lost one or more virulenceplasmids. In specific embodiments, the strain is wild-type Y. pestisKIM6, although any number of the modified KIM6 strains may be employed(e.g., KIM6 del PhoP (regulator) could be made with LpxF+(expressing aphosphatase) or KIM6 del LpxD (acyltransferase) could be made with a delPmrK (which would not add aminoarabinose).

In one embodiment, there is a method of generating alipooligosaccharide/lipid A-based mimetic, comprising the steps ofobtaining a bacterial strain that has one or more of the followingmodifications: expresses one or more non-endogenous lipid A biosynthesisenzymes; expresses one or more endogenous lipid biosynthesis enzymes,wherein the enzyme is modified; and/or has modified regulation of one ormore endogenous lipid biosynthesis enzymes; and subjecting the strain toconditions suitable for production of the lipooligosaccharide/lipid Acomposition. In particular embodiments, the obtaining step is furtherdefined as engineering the bacterial strain to have one or more of themodifications. In some cases, the engineering step comprises one or moreof delivering a vector into the bacteria; and/or bacterial conjugation.In specific embodiments, the engineering step comprises delivering avector into the bacteria, wherein the vector comprises sequence thatencodes one or more non-endogenous lipid A biosynthesis enzymes. Inspecific embodiments, the one or more non-endogenous lipid Abiosynthesis enzymes is an acyltransferase, deacylase, phosphatase,glycosyltransferase, or a mixture thereof, from another bacterialstrain. In particular embodiments, the engineering step comprisesmodifying the bacteria to express a modified endogenous lipidbiosynthesis enzyme. In some cases, the modified endogenous lipidbiosynthesis enzyme comprises a mutation in the enzyme. In certainembodiments, the one or more modified endogenous lipid biosynthesisenzymes is an acyltransferase, deacylase, phosphatase, orglycosyltransferase. In some cases, the engineering step comprisesmodifying the bacteria to have modified regulation of expression of oneor more endogenous lipid A biosynthesis enzymes. In specificembodiments, modifying the bacteria to have modified regulation ofexpression of one or more endogenous lipid biosynthesis enzymes isfurther defined as mutating a gene in the bacteria that is a regulatorygene for lipid A biosynthesis in the bacteria, including one defined asa sensor kinase, a response regulator, or both of a two-componentregulatory system in the bacteria. In certain cases, the regulatory genefor lipid A biosynthesis is histidine kinase. In some cases, the one ormore endogenous lipid biosynthesis enzymes is an acyltransferase,deacylase, phosphatase, or glycosyltransferase. In particularembodiments, the subjecting step comprises particular temperatureconditions suitable for production of the lipooligosaccharide/lipid Acomposition.

In certain embodiments of methods of generating alipooligosaccharide/lipid A-based mimetic, the lipooligosaccharide/lipidA-based mimetic produced by the method has a modified level and/orcontent of fatty acid compared to the endogenous bacterial lipid Amolecule or a reference lipid A molecule. In particular embodiments, thelipooligosaccharide/lipid A-based mimetic produced by the method has amodified number of phosphates compared to the endogenous bacterial lipidA molecule or a reference lipid A molecule. In certain embodiments, thelipooligosaccharide/lipid A-based mimetic produced by the method hasmodified phosphates compared to the endogenous bacterial lipid Amolecule or a reference lipid A molecule. In particular aspect, themodified phosphates are further defined as having only one sugar (simpleand aminosugars) or having an additional sugar linked to the phosphateor ethanolamine. In specific aspects, the lipooligosaccharide/lipidA-based mimetic produced by the method has a modified sugar numberand/or content compared to the endogenous bacterial lipid A molecule ora reference lipid A molecule. In some cases, thelipooligosaccharide/lipid A-based mimetic comprises one or three or moresugars. The lipooligosaccharide/lipid A-based mimetic may comprisemodified sugars selected from the group consisting of aminoarabinose,glucosamine, and galactosamine. In some cases, the method furthercomprises the step of analyzing extracts from the bacteria for thestructure, function, or both of the lipooligosaccharide/lipid A-basedmimetic.

In specific aspects, the analyzing step comprises analyzing thestructure of the lipooligosaccharide/lipid A-based mimetic by performingone or more types of mass spectrometry, gas chromatography, or acombination thereof. In some cases, the analyzing step comprisesanalyzing the function of the lipooligosaccharide/lipid A-based mimeticby measuring an inflammatory response of the lipooligosaccharide/lipidA-based mimetic. In particular embodiments, the inflammatory response isa proinflammatory response, such as one measured by cell stimulation ofmacrophages. The proinflammatory response may be measured by activationof cell-mediated pathways, humoral pathways, or both.

In some embodiments of methods of generating a lipooligosaccharide/lipidA-based mimetic, the bacterial strain is Yersinia pestis, Pseudomonas,an Archaebacteria or an extremophile. In specific embodiments, the oneor more non-endogenous lipid A biosynthesis enzymes is from Pseudomonasaeruginosa, Francisella novicida, E. coli, Bordetella subspecies,Helicobacter pylori, or Salmonella typhimurium. In some aspects of themethod, the method further comprises the step of combining thelipooligosaccharide/lipid A-based mimetic with an antibody, a weakenedmicrobe, a killed microbe, one or more antigens, a toxoid,polysaccharide, or nucleic acid to produce an immunogenic composition.In some cases, the method further comprises the step of delivering aneffective amount of the immunogenic composition to an individual in needthereof.

In some embodiments, the composition is a compound of formula (I)

wherein R₁ and R₂ may be H, OH, protonated phosphate, a phosphate salt,a sugar phosphonate, or a mono-, di- or poly-saccharide, R₃ may be OH ora mono-, di- or poly-saccharide, R₄, R₅, R₆ and R₇ may be an alkyl oralkenyl chain of up to 13 carbons (for a chain of 16 carbons), and R₈,R₉, R₁₀ and R₁₁ may be H, OH, or an alkyl or alkenyl ester of up to 16carbons.

In some embodiments, there is provided a biosynthetic, immunomodulatinglipid polysaccharide compound of formula (I)

wherein R₁ and R₂ may be H, OH, protonated phosphate, a phosphate salt,a sugar phosphonate, or a mono-, di- or poly-saccharide, R₃ may be OH ora mono-, di- or poly-saccharide, R₄, R₅, R₆ and R₇ may be an alkyl oralkenyl chain of up to 13 carbons, and R₈, R₉, R₁₀ and R₁₁ may be H, OH,or an alkyl or alkenyl ester of up to 18 carbons.

In one embodiment, there is a method of enhancing an immune response ina subject, comprising administering to the subject an effective amountof an immunogenic compound of formula (I)

wherein R₁ and R₂ may be H, OH, protonated phosphate, a phosphate salt,a sugar phosphate, or a mono-, di- or poly-saccharide, R₃ may be OH or amono-, di- or poly-saccharide, R₄, R₅, R₆ and R₇ may be an alkyl oralkenyl chain of up to 13 carbons, and R₈, R₉, R₁₀ and R₁₁ may be H, OH,or an alkyl or alkenyl ester of up to 16 carbons.

In some embodiments, a biosynthetic, immunomodulating lipidpolysaccharide compound is at least one of:

where the integers adjacent to fatty acid chains represent the number ofcarbons atoms in the respective fatty acid chain, and the ratiosadjacent to fatty acid chains represent the number of carbons atoms inthe respective fatty acid chain and the number of olefins within thefatty acid chain.

In particular embodiments, there is a kit, comprising one or morecompositions of the disclosure. In specific embodiments, the kit furthercomprising one or both of an antigen and a pharmaceutically acceptablecarrier.

It should be understood that the specific compositions and methodsdescribed herein are included by way of example and should not beunderstood as limiting. In consideration of the teachings herein, onehaving ordinary skill in the art would be able to develop additionalcompounds and methods of interest. Such compounds do not deviate fromthe overall spirit and therefore should be considered part of thisinvention.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawing, in which:

FIG. 1 shows representative Yp-based lipid A structures generated usingBECC, as compared to WT grown at 37° C. (mammalian host temperature).X-potential addition site for aminoarabinose.

Presence or absence of a 3-OH C14 fatty acid representingmicroheterogeneity of 3 position fatty acid group due to hydrolysis ofLPS during MPL synthesis.

FIG. 2 demonstrates that a modified Yp LOS is immunostimulatory.Purified LOS was tested by an in vitro cell stimulation assay usingdifferentiated (Vitamin D₃) THP-1 cells. Dose-response stimulations withLOS for 24 hours, supernatants were assayed by ELISA for IL-8 and humanRANTES (R&D Sys.). Re595 response at 100 ng/mL, a potent TLR4 agonistpositive control; +37 C, illustrates potency of rational modification ofa non-stimulatory (WT) structure. GlaxoSmithKline (GSK) and Avanti MPL(slightly different lipid A structures), response at 1 μg/ml for RANTESis 641 pg/ml and 147 pg/ml, respectively. No response was observed at100 μg/ml.

FIG. 3 shows that modified LOS enhances adaptive immune response toprotein antigen. C57BL/6 mice were dosed, IP, with F1 protein antigen incombination with possible adjuvant candidate ΔphoP 37° C., boosted at 4weeks, serum was harvested 1 week post-boos and IgG subtypes tittered byELISA. Dosing regimen used; 25 μg Yp F1+/−50 μg CpG, Avanti MPL, andΔphoP LOS.

FIGS. 4A-4E illustrate representative Yp-based lipid A structures (FIGS.4B-4C) generated using BECC, as compared to WT Yp grown at 37° C. (FIG.4A), AGP (FIG. 4D), and MPL (FIG. 4E). Structures in FIG. 4B and FIG. 4Chave been modified through in trans BECC expression of theacyltransferases—PagP, MsbB, and LpxP. TLR4 mimetics (FIG. 4D)aminoalkyl glucosaminide phosphates (AGP), pink star indicates 1position phosphate moiety is replaced by carboxylic acid and (FIG. 4E)monophosphorylated lipid A (MPL)—GSK, blue bracket representheterogeneity of fatty acid number from 3-7 fatty acids present onglucosamine backbone and green star indicates removal of 1 positionphosphate moiety in the GSK MPL preparations. Note: Avanti MPL is asynthetically derived hexa-acylated lipid A with all fatty acids 14carbons in length. Lipid A modifications in these strains (FIGS. 4A-4C)have been confirmed by MS and GC analysis.

FIG. 5 shows that modified Yp LOS is immunostimulatory. Purified LOS wastested by an in vitro cell stimulation assay using differentiated(Vitamin D3) THP-1 cells. Dose-response stimulations with LOS for 24hours, supernatants were assayed by ELISA for IL-8, human RANTES, andIL-23. Re595, a potent TLR4 agonist was used as a positive control at100 ng/mL; pagP+37° C. LOS illustrates potency of rational modificationof a non-stimulatory (WT) structure. MPL obtained from GSK and Avanti(slightly different lipid A structures) response at 1 mg/ml for RANTESis 641 pg/ml and 147 pg/ml, respectively. No response was observed at100 μg/ml.

FIG. 6. Provides that modified LOS enhances adaptive immune response toprotein antigen. C57BL/6 mice were dosed, IP with F1 protein antigen incombination with possible adjuvant candidate ΔphoP 37° C., boosted at 4weeks, serum was harvested 1 week post-boost and IgG1 titered by ELISA.Dosing regimen used: 25 μg Yp F1 antigen; 50 μg CpG, MPL, or Yp ΔphoPLOS.

FIG. 7 shows examples of pathways of adjuvant outcomes for TLR4activation.

FIG. 8 provides a description and heterologous sources of LOS/Lipid Amodifying enzymes for BECC, illustrating examples of modifying enzymesthat may be used to generate Y. pestis or other strains with modifiedLOS.

FIG. 9 provides a description of individual strains generated, structureof the resultant lipid A, and mass at 37° C.

FIG. 10 provides description of individual strains generated, structureof the resultant lipid A, and mass when the Y. pestis strain was grownat 26° C.

FIG. 11 shows proinflammatory responses of specific glycolipidstructures—Human cell lines when the Y. pestis strain was grown at 37°C. N/A: not available.

FIG. 12 provides proinflammatory responses of specific glycolipidstructures—Human cell lines when the Y. pestis strain was grown at 26°C.

FIG. 13 demonstrates the structure of exemplary LPS molecules generatedby methods of the disclosure.

FIG. 14 demonstrates the structure of exemplary LPS molecules generatedby methods of the disclosure.

FIG. 15 demonstrates the structure of exemplary LPS molecules generatedby methods of the disclosure.

FIG. 16 demonstrates the structure of exemplary LPS molecules generatedby methods of the disclosure.

FIG. 17 demonstrates the structure of exemplary LPS molecules generatedby methods of the disclosure.

FIG. 18 demonstrates the structure of exemplary LPS molecules generatedby methods of the disclosure.

FIG. 19 demonstrates the structure of exemplary LPS molecules generatedby methods of the disclosure.

FIG. 20 demonstrates the structure of an exemplary LPS moleculegenerated by methods of the disclosure.

FIG. 21 illustrates the structure of Control LPS molecules.

DETAILED DESCRIPTION OF THE INVENTION

In keeping with long-standing patent law convention, the words “a” and“an” when used in the present specification in concert with the wordcomprising, including the claims, denote “one or more”. Some embodimentsmay consist of or consist essentially of one or more elements, methodsteps, and/or methods of the disclosure. It is contemplated that anymethod or composition described herein can be implemented with respectto any other method or composition described herein embodiments that aredisclosed and still obtain a like or similar result without departingfrom the spirit and scope of the subject matter.

The term “lipooligosaccharide/lipid A mimetic” as used herein refers tocompound(s) that act on Toll-like receptor 4 (TLR4) and haveimmunomodulatory effects when used both as vaccine adjuvants or asstand-alone products.

Most vaccines contain adjuvants to help stimulate the production ofimmunity against the antigens or to stabilize antigen formulations toincrease immune composition efficacy, including vaccine efficacy.Currently, there are few adjuvants licensed for use in human vaccines.These include alum salts, oil in water emulsions, the chemically derivedmonophosphoryl lipid A (MPL), a non-toxic toll-like 4 (TLR4) agonist. Anumber of synthetic lipid A mimetics (aminoalkyl glucosaminide4-phosphates—AGPs) are presently under evaluation in bacterial and viralanimal infection models. However, because of the variable heterogeneityfrom the chemical hydrolysis of LPS for the production of MPL and thelimitations and labor-intensive nature of synthesizing AGPs that moreclosely mimic the structure of lipid A, alternative technologies arerequired for identifying novel adjuvants.

I. General Embodiments

Embodiments of the disclosure include lipooligosaccharide/lipid A-basedmimetic compositions produced by bacteria for use in immunogeniccompositions, such as for use as adjuvants, and the disclosure alsoincludes methods of making and using the compositions. The compositionsin certain aspects are lipid A mimetics, and they may be used alone asan immunogenic composition or with another immunogenic composition toelicit a desired immune response in an individual. In specificembodiments, the compositions are useful to block or arrest adverse invivo immunological response, such as endotoxic shock by altering bindingto TLR4. Embodiments include methods of generating the compositions, andfurther steps of the methods employ use of the compositions foradministering to a mammal for eliciting an immune response.

In particular embodiments, this disclosure establishes processes for thesynthesis and characterization of novel TLR4 mimetics with definedLOS/lipid A modifications utilizing novel bacterial enzymaticcombinatorial chemistries (BECC) that will be useful to “fine tune” thebalance between activating immunity while avoiding excessive adversehost reactions, such as inflammation. The in vitro and in vivo studiesdescribed herein establish a novel mechanism for adjuvant/vaccine designleading to well-defined, cell-type specific adjuvants that can targethost cell-mediated responses plus robust antibody production.Ultimately, the future of vaccinology, particularly with regards tointractable infections and insufficient vaccine protection, isfundamentally linked to the development of safe, efficacious, andreliable adjuvants, and the present disclosure addresses this need.

Generation of novel, rationally-designed adjuvants normally requires themultidisciplinary effort in the fields of immunology, biochemistry,microbiology, and pharmacology, and the process of chemically derivingor synthesizing these molecules is expensive, labor intensive, andcomplex. In an aspect of this disclosure, one can “harness” the normalbacterial lipid A biosynthesis pathways present in all Gram-negativebacteria to synthesize structures based on the presence or absence ofspecific phosphate, acyl, and carbohydrate groups (for example). Thesestructures can be produced efficiently, rapidly, and in sufficientquantities for use as stand-alone immunotherapeutic molecules oradjuvants in new immunogenic compositions, including vaccineformulations, for example. Thus, the bacteria utilized in the methods ofthe disclosure act as factories to supersede the extensivemultidisciplinary efforts normally used to synthesize desired adjuvantmolecules.

II. Exemplary Adjuvant Compositions

In particular embodiments, one or more adjuvant compositions aredisclosed herein. One or more adjuvant compositions may be generated bymethods of the invention, although one or more adjuvant compositions maybe generated by other means and are used in methods of use in thedisclosure, or one or more adjuvant compositions may be present innature and are used in methods of use in the disclosure. Thecompositions may be provided to an individual in need thereof in aneffective amount to produce an immune response in the individual that ispotent, but not cytotoxic.

In certain embodiments of the disclosure, lipooligosaccharidescomprising an oligosaccharide core with covalently-attached fatty acidchains are provided. The oligosaccharide sugars may be linked throughO-, N-, S- or C-glycosidic bonds. The glycosidic bonds may be (1-4′) or(1-6′), α- or β-glycosidic bonds. In particular embodiments, thelipooligosaccharides comprise a β(1-6′)-D-glucosamine disaccharide core.The oligosaccharide sugars may be D-sugars, L-sugars, or a mixturethereof. In some embodiments, the covalently-attached fatty acid chainsare alkyl fatty acids. In other embodiments, covalently-attached fattyacid chains may include at least one olefin, which may be a cis- ortrans-olefin, or a mixture thereof, and/or at least one hydroxyl group.In certain aspects, a fatty acid chain may be covalently attached to thefatty acid hydroxyl group. The fatty acid chains may comprise continuouscarbon chains of up to 18 carbon atoms. More preferable fatty acidscomprise continuous carbon chains of 12, 14 or 16 carbon atoms. Thefatty acid chains may be covalently attached to the oligosaccharide corethrough a variety of chemical bonds, including, but not limited to,ester, amide and thioester bonds. The modified lipooligosaccharides mayinclude at least one phosphate group or sugar-phosphate ester. A carbonatom bearing a phosphate group or sugar-phosphate ester may be in an Ror S stereochemical configuration. The sugar-phosphate ester maycomprise a mono-, di- or poly-saccharide covalently attached to thephosphate group.

Thus, particular compositions of the disclosure comprise multiplemoieties, and one or more of the moieties may be modifiable, includingmodifiable methods of the disclosure. In particular embodiments, thecompositions comprise at least a sugar moiety, a phosphate moiety and afatty acid moiety. Compositions of the disclosure may differ in one ormore of the moieties.

A. Sugar Moiety(ies)

In specific embodiments, the compositions comprise one or more sugarunits with attached acyl chains. In certain aspects, the compositionscomprise a disaccharide. Although in Lipid A the sugars contain onephosphate group on each sugar, the lipid A mimetics of the disclosuremay contain more than one or two phosphate group or no phosphate groups.In specific cases, the compositions comprise one, two, or three sugars,although more sugars may be included. The sugars may be of any kind, butin specific embodiments the simple sugars are glucose, galactose,arabinose, fructose, ribose, or the aminosugars are aminoarabinose,glucosamine, galactosamine, and so forth. In particular embodiments, thesugars have a linkage group to the fatty acids, such as an amine group,ketone group, and so forth.

B. Phosphate Moiety(ies)

In particular embodiments, the compositions comprise at least onephosphate, including one phosphate, two phosphates, or three or morephosphates. Each sugar in the molecule may have one or more phosphates.Each phosphate in the composition may be unmodified or may be modifiedto have attached thereto another group, such as another sugar as definedabove or ethanolamine.

C. Fatty Acid Moiety(ies)

In certain embodiments, the compositions comprise at least one fattyacid moiety. In some cases the compositions comprise one, two, three,four, five, six or more fatty acids. In particular embodiments, no morethan eight fatty acids are included in the compositions. The length ofone or more of the fatty acid chains may be an aspect for modification.In specific cases all of the fatty acids are of the same length, whereasin other cases the fatty acids may be of different lengths. In certainmolecules, all but one fatty acid are of the same length. In specificembodiments, one or more fatty acids are up to twenty-two carbons inlength. Specifically, the fatty acids may be 10, 12, 14, 16, 18, 20, 22,or more carbons in length. In particular embodiments, the fatty acidshave at least one double bond, including one double bond, two doublebonds, three double bonds, and so forth.

III. Extraction of Lipooligosaccharide/Lipid A-Based Mimetics

Upon production of the lipooligosaccharide/lipid A-based mimetics in theselected bacterial strain, the lipooligosaccharide/lipid A-basedmimetics are obtained from the bacteria. The mimetic molecules may beobtained by any suitable method, but in specific embodiments they arechemically extracted using standard LOS extraction protocols. Inspecific cases, initially multiple types of LOS extraction proceduresare employed to obtain LOS from the bacteria, and extraction proceduresmay be performed more than once. In particular aspects, the extractionprocedures are phenol-based, magnesium-precipitation-based, ammoniumhydroxide/isobutyric acid-based, chloroform-methanol-based,detergent-based, and so forth.

Once the LOS preparation is obtained from the bacteria, the lipid Afraction is liberated using gentle hydrolysis to protect sensitivestructural elements.

In particular embodiments, lipid A or its mimetics may be isolated asfollows. Lipid A was isolated after hydrolysis in 1% SDS at pH 4.5.Briefly, 500 μl of 1% SDS in 10 mM Na-acetate (pH 4.5) was added to alyophilized sample. Samples were incubated at 100° C. for 1 h, frozen,and lyophilized. The dried pellets were resuspended in 100 μl of waterand 1 ml of acidified ethanol (100 μl 4 N HCl in 20 ml 95% ethanol).Samples were centrifuged at 5,000 rpm for 5 min. The lipid A pellet wasfurther washed (three times) in 1 ml of 95% ethanol. The entire seriesof washes was repeated twice. Samples were resuspended in 500 μl ofwater, frozen on dry ice, and lyophilized. Lipid A was used formatrix-assisted laser desorption ionization (MALDI) mass spectrometryanalysis.

IV. Analysis of Exemplary Lipooligosaccharide/Lipid A-based MimeticCompositions

Following extraction of the desired lipooligosaccharide/lipid A-basedmimetic, the mimetic is analyzed for structure and/or function. Incertain cases, the structure is analyzed prior to the function, whereasin other cases the function is analyzed prior to the structure. Theanalysis may be on a small scale with only a few mimetics being analyzedat substantially the same time or on a large scale with many mimeticsbeing analyzed at substantially the same time.

In some cases, the structure is analyzed by routine methods in the art,including using one or more procedures for the analysis. In particularembodiments, mass spectrometry, gas chromatography, or both are utilizedfor analysis of structure. For mass spectrometry embodiments,matrix-assisted laser desorption/ionization/time-of-flight massspectrometry (MALDI-TOF) and electrospray ionization (ESI) are utilized,including in both the negative and positive-ion mode. Other steps toanalyze LOS/lipid A fatty acid content can include acid hydrolysis,methylation, and hexane extraction.

In specific embodiments, one or more structures produced by methods ofthe disclosure are analyzed as follows.

V. Electrospray Ionization Linear Ion Trap Fourier Transform IonCyclotron Resonance Mass Spectrometry

Lipid A was analyzed by ESI in the negative mode of an LTQ-FT linear iontrap Fourier transform ion cyclotron resonance mass spectrometer (ThermoFisher). Samples were diluted to ˜0.3-1.0 mg/ml in chloroform/methanol(1:1) and infused at a rate of 0.5-1.0 ul/min via a fused silicacapillary (75 um i.d./360 um o.d.) with an ˜15 um spray tip (NewObjective). Instrument calibration and tuning parameters were optimizedby using a solution of Ultramark 1621 (Lancaster Pharmaceuticals). Forexperiment acquired in the ICR cell, resolution was set at 100K and ionpopulations were held constant by automatic gain control at 1.0×10⁶ and5.0×10⁵ for MS and MS/MS, respectively. For tandem mass spectra, theprecursor ion selection window was set to 4-8 DS and the collisionenergy was set to 30% on the instrument scale. The CID MS' analysis inthe linear ion trap was acquired with an ion population of 1.0×10⁴maximum fill time of 200 ms. The subsequent MS³ and MS⁴ had an isolationwindow of 2 Da with a collision energy of 25%. All spectra were acquiredover a period of 1-2 min and averaged. Typically, MS and MS² events weremass analyzed in the ICR cell, and the MS³ and MS⁴ were mass analyzed inthe LTQ. Infrared multiphoton dissociation (IRMPD) MS²events wereacquired in the ICR cell using similar detection parameters to thosedescribed above. Precursor ions were irradiated by IR photons producedby a CO₂ laser [Synrad firestar Series V20), Model FSV20SFB; 75 W(10.2-10.8 um)] with pulse durations of 20-100 ms and pulse power of20-80%. Data were acquired and processed with Xcaliber (version 1.4;Thermo Fisher) using seven-point Gaussian smoothing. On-line liquidchromatography ESI tandem MS experiments were performed by interfacing acustom-fabricated microcolumn (fused-silica capillary) packed withsilica to the LTQ-FTESI source.

VI. Electrospray Ionization Tandem Quadrupole Mass Spectrometry

Lipid A was analyzed by ESI in the negative ion mode on a Sciex API IIItandem quadrupole mass spectrometer (Perkin Elmer). Samples were dilutedto ˜0.3-1.0 mg/ml in chloroform/methanol (1:1) and infused at a rate of0.5-1.0 ul/min via a fused silica capillary (i.d. 100 um) by using asyringe pump (Harvard Apparatus Model 11). The instrument was operatedwith the following settings: needle voltage, −4300 V; counter electrode,−650 V, nebulizer gas pressure, 20 psi; curtain gas pressure, 10 psi;declustering potential, −35 V; collision cell entrance potential, −10 V;collision cell exit potential, −15V; and collision gas, argon. Tandem MSdata were acquired in both product and precursor ion scan modes.

VI. MALDI-TOF MS Analysis

Lipid A structures were assessed by negative-ion MALDI-TOF MS.Lyophilized lipid A was extracted in chloroform/methanol and then 1 μlwas mixed with 1 μl of Norharmane MALDI matrix. All MALDI-TOFexperiments were performed using a Bruker Autoflex Speed MALDI-TOF massspectrometer (Bruker Daltonics, Billerica, Mass.). Each spectrum was anaverage of 300 shots. ES tuning mix (Aligent, Palo Alto, Calif.) wasused for calibration.

VII. Gas Chromatography

LPS fatty acids were converted to fatty acid methyl esters and analyzedby gas chromatography (GC) essentially as previously described (ref).Briefly, 10 mg of lyophilized bacterial cell pellet was incubated at 70°C. for 1 hour in 500 μl of 90% phenol and 500 μl of water. Samples werethen cooled on ice for 5 minutes and centrifuged at 10,000 rpm for 10minutes. The aqueous layer was collected and 500 μl of water was addedto the lower (organic) layer and incubated again. This process wasrepeated twice more and all aqueous layers were pooled. Two ml of ethylether was added to the harvested aqueous layers, this mixture was thenvortexed and centrifuged at 3,000 rpm for 5 minutes. The lower (organic)phase was then collected and 2 ml of ether were added back remainingaqueous phase. This process was carried out twice more. The collectedorganic layer was then frozen and lyophilized overnight. LPS fatty acidswere converted to fatty methyl esters, in the presence of 10 μgpentadeconic acid (Sigma, St Louis, Mo.) as an internal standard, with 2M methanolic HCl (Alltech, Lexington, Ky.) at 90° C. for 18 hours.

Functional analysis of the lipid A mimetic structures may be performedby standard methods in the art to ascertain immune function of themimetics. In vitro analysis methods may be preceded by in vivo analysismethods. In at least some cases, output of the methods for theparticular mimetic is compared to a reference, such as MPL, for example.Those mimetic compositions having suitable immune function may beutilized as an adjuvant. In specific aspects, the desired molecule haslow toxicity yet is immunostimulatory as shown, for example, bytriggering chemotaxis without profound apoptosis or pyroptosis. Examplesof in vitro methods to analyze the function of the molecules includes atleast an in vitro cell stimulation assay, and a range of doses of theparticular lipid A mimetic being tested may be screened. The cells forthe cell stimulation may be of a human monocytic cell line, for example.The stimulation of the cells may be measured by assaying for certaincytokines (such as IL-8) and by assaying for RANTES (regulated onactivation, normal T cell expressed and secreted), which is a chemokine.Those candidate molecules that show utility in the in vitro aspects maybe tested in the in vivo assays. The candidate lipooligosaccharide/lipidA-based mimetics may be assayed by in vivo models, such as in vivomurine screening models. In specific aspects, the candidate moleculesare provided to mice alone and with known immunogenic reagents to probeinnate and adaptive immune potentiation; in certain aspects, directmodulation of cell mediated and humoral immunity and balancing ofTh₁/Th₂ responses are examined.

In specific embodiments, the function of the compositions may be assayedas follows.

Primary screening will be performed for all new molecules in systemicand airway mouse and human macrophage cell lines (RAW, THP-1, MH-S,U937, respectively) over a wide dose range. Supernatants from cellstimulations will be screened by cytokine multiplex assays forinflammatory markers of macrophage activation, including IL-1β. Primaryscreening will be performed for all new molecules in systemic and airwaymouse and human macrophage cell lines (RAW, THP-1, MH-S, U937,respectively) over a wide dose range. Supernatants from cellstimulations will be screened by cytokine multipleest for development ofan adjuvant molecule include down-modulated inflammatory cytokineprofiles (low IL-8) and up-regulated T cell fate-determining cytokines,such as IL-12 (promotes T_(H)1) and IL-23p19 (promotes T_(H)17).Tertiary screening will verify TLR4-mediated activity of the candidatemolecules using human and mouse TLR4-transfected HEK cells with aninflammatory activation (NF-κB). Tertiary screening will verifyTLR4-mediated activity of the candidate molecules using human and mouseTLR4-transfected HEK cells with an inflammatory activation (NF-se range.Supernatants from cell stimulations will be screened by cytokinemultiple BECC-synthesized TAMs, immature DCs from mouse and human (BMDC,PBMC) will be stimulated and analyzed for maturation (CD83) andactivation (CD86, MHCII, etc.) surface markers by flow cytometry.

Finally, ten candidates will be identified from the outlined in vitroscreening to advance to an in vivo screening for acute toxicity. C57BL/6mice (n=5 per group) will receive a single, intraperitoneal (IP) dose ofa candidate molecule. Animals will be monitored closely for clinicalsigns of acute toxicity and/or a lethal inflammatory response includingloss of weight, core temperature dysregulation, and behavioral changes.Sera will be collected and assayed for unacceptably high circulatingendotoxemia markers (TNF-α). Sera will be collected and assayed forunacceptably high circulati:creatinine ratio, ALT, AST, IRN, totalprotein) 1-7 days post-injection.

In particular embodiments, compositions of the disclosure are assayed inchallenge studies. The specific challenge models employed will be thosethat will differentiate between candidate compound outcomes. Asexamples, the S. aureus dermonecrosis model for the generation ofhigh-affinity anti-A-T mAbs may be employed. Here, mice are immunized 3×biweekly via the intramuscular route, then intradermally challenged witha dose of S. aureus calibrated to induce non-healing dermal lesion.Monitoring of lesion size are conducted in vaccinated mice, and skinbiopsies are analyzed for presence of cytokines, bacterial load, andinfiltrating leukocytes. Alternatively, a Respiratory syncytial virus(RSV) infection model in mice may be utilized if candidate compoundsdemonstrate superior capability in the induction of RSV-neutralizing Absand T cell responses.

VIII. Use of Exemplary Lipooligosaccharide/Lipid A-Based MimeticCompositions

The compositions of the present disclosure may be used for medicinalpurposes of any kind, but in specific embodiments, the lipid A mimeticcompositions are employed as an immunogenic composition alone or as partof an immunogenic composition having at least one component other thanthe lipid A mimetic. The immunogenic composition may be an adjuvant, forexample. Adjuvants are useful because they reduce the amount of theactive component required in an immunogenic composition, they may allowthe use of fewer doses of the immunogenic composition because the immuneresponse is stronger and lasts longer, and those with compromised immunesystems benefit from them because their immune system requires an extraboost to provide protection.

In particular cases, more than one lipid A mimetic is employed in animmunogenic composition, including two, three, or more lipid A mimetics.The lipid A mimetic may be formulated for use as an immunogeniccomposition, including having an appropriate carrier excipient. Atherapeutically effective amount of the composition(s) is employed, andthe proper amount may be determined by any suitable method in the art.The immunogenic composition may be delivered to the individual by anyappropriate means, including by injection or orally. Multiple deliveriesof the lipooligosaccharide/lipid A-based mimetic(s) may be employed.

The delivery of the lipid A mimetic in an immunogenic composition mayoccur prior to exposure of the individual to a pathogen and/or followingexposure of the individual to a pathogen. In some cases, the lipid Amimetic-comprising immunogenic composition is given to an individual aspart of a routine medical practice in preventative measures. In certaincases, the lipid A mimetic immunogenic composition is delivered to anindividual in anticipation of exposure to a pathogen or in anticipationof being at risk for exposure to a pathogen. In some cases, theimmunogenic composition is provided to an individual not for use with apathogen, but instead is used for another medical condition, such as forcancer or inflammation-mediated coronary heart disease, for example.Thus, the immunogenic composition may be prophylactic or therapeutic.

In some cases, the lipid A mimetic composition is employed for immunestimulation for a pathogen, such as any bacteria or virus. However, inspecific embodiments, the immunogenic composition of the disclosure isuseful for measles, rubella, mumps, yellow fever, typhoid fever,smallpox, polio, tuberculosis, plague, chickenpox, human papilloma virus(HPV), influenza, Haemophilus influenzae type B, rotavirus, pneumonia,hepatitis A, hepatitis B, hepatitis C, diphtheria, pertussis, tetanus,meningococcal, West Nile Virus, Dengue, Japanese encephalitis virus,Chikungunya, HIV, tularemia, salmonellosis, listeriosis, and/orShigella. The lipid A mimetic immunogenic compositions may be applied tomammals other than humans, such as dogs, cats, horses, cows, pigs,sheep, and so forth. For example, a dog may be given an immunogeniccomposition comprising a lipid A mimetic of the disclosure, such as animmunogenic composition for canine distemper, canine parvovirus,infectious canine hepatitis, adenovirus-2, leptospirosis, Bordetella,canine parainfluenza virus, and Lyme disease, for example.

In some cases, a lipid A mimetic(s)-comprising immunogenic compositioncomprises a plurality of antigenic compositions each suitable for adifferent pathogens, thereby being prophylactic for more than onepathogen. In such cases, one or more lipid A mimetics may be employed.

VIX. Vaccines and Immunogenic Compositions Generally

Embodiments of the disclosure employ lipooligosaccharide/lipid A-basedmimetics as adjuvants either alone or with another immunogeniccomposition. In some embodiments, the immunogenic composition to beemployed with the lipooligosaccharide/lipid A composition is a vaccine.The immunogenic composition (which may be referred to as an adjuvanticcomposition of the disclosure may be considered an antigeniccomposition. For an immunogenic composition to be useful, it must inducean immune response to the antigen in a cell, tissue or animal (e.g., ahuman). As used herein, an “antigenic composition” may comprise anantigen (e.g., a peptide, polypeptide, weakened microbe, a killedmicrobe, one or more antigens, a toxoid, polysaccharide, or nucleicacid), a nucleic acid encoding an antigen (e.g., an antigen expressionvector), or a cell expressing or presenting an antigen. In otherembodiments, the immunogenic composition is in a mixture that comprisesan additional immunostimulatory agent or nucleic acids encoding such anagent. Immunostimulatory agents include but are not limited to anadditional antigen, an immunomodulator, an antigen presenting cell, oran adjuvant that is other than the lipid A mimetics of the disclosure.In other embodiments, one or more of the additional agent(s) iscovalently bonded to the antigen or an immunostimulatory agent, in anycombination. In certain embodiments, the antigenic composition isconjugated to or comprises an HLA anchor motif amino acids.

In certain embodiments, an antigenic composition or immunologicallyfunctional equivalent comprising the lipid A mimetic of the disclosuremay be used as an effective vaccine in inducing a humoral and/orcell-mediated immune response in an animal. The present disclosurecontemplates using a lipid A mimetic in one or more immunogeniccompositions or vaccines for use in both active and passive immunizationembodiments.

In some cases, one or more immunogenic composition components may becomprised in a lipid or liposome. In another non-limiting example, theimmunogenic composition may comprise one or more adjuvants. Animmunogenic composition may be prepared and/or administered by anymethod disclosed herein or as would be known to one of ordinary skill inthe art, in light of the present disclosure.

The type of antigens that may be employed with one or more lipid Amimetics of the disclosure may be of any kind, including a proteinaceousantigen that may be produced by chemical synthesis or expression from anucleic acid sequence, for example. Proteinaceous antigens may comprisea peptide or polypeptide. Another type of antigen in which a lipid Amimetic is used as an adjuvant includes genetic antigens, wherein animmune response may be promoted by transfecting or inoculating an animalwith a nucleic acid encoding an antigen, following which one or morecells comprised within a target animal then expresses the sequencesencoded by the nucleic acid after administration of the nucleic acid tothe animal; the antigen may also be in the form, for example, of anucleic acid (e.g., a cDNA or an RNA) encoding all or part of thepeptide or polypeptide sequence of an antigen. Expression in vivo by thenucleic acid may be, for example, by a plasmid type vector, a viralvector, or a viral/plasmid construct vector. In yet another case, alipid A mimetic is employed with a cellular antigen comprising a cellexpressing the antigen, such as a cell isolated from a culture, tissue,organ or organism. The cell may be transfected with a nucleic acidencoding an antigen to enhance its expression of the antigen. Of course,the cell may also express one or more additional components, such asimmunomodulators or adjuvants (other than the lipid A mimetic adjuvant).An immunogenic composition may comprise all or part of the cell.

It is contemplated that an antigenic composition includes the antigenand the lipooligosaccharide/lipid A-based mimetic(s), but there may alsobe one or more additional components to form a more effective antigeniccomposition. Non-limiting examples of additional components include, forexample, one or more additional antigens, immunomodulators or adjuvantsto stimulate an immune response to the antigenic composition.

For example, it is contemplated that immunomodulators can be included inthe composition to augment a cell's or a patient's (e.g., an animal's)response. Immunomodulators can be included as purified proteins, nucleicacids encoding immunomodulators, and/or cells that expressimmunomodulators in the vaccine composition. In specific embodiments,various combinations of immunomodulators may be used (e.g., a cytokineand a chemokine). When cytokines are included in the compositions, theymay be interleukins, cytokines, nucleic acids encoding interleukins orcytokines, and/or cells expressing such compounds are contemplated aspossible vaccine components. Interleukins and cytokines include but arenot limited to interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-18, IL-22,IL-23 β-interferon, α-interferon, g-interferon, angiostatin,thrombospondin, endostatin, GM-CSF, G-CSF, M-CSF, METH-1, METH-2, tumornecrosis factor, TGFβ, LT and combinations thereof. In some cases,chemokines, nucleic acids that encode for chemokines, and/or cells thatexpress such also may be used as components of the composition.Chemokines generally act as chemoattractants to recruit immune effectorcells to the site of chemokine expression. It may be advantageous toexpress a particular chemokine coding sequence in combination with, forexample, a cytokine coding sequence, to enhance the recruitment of otherimmune system components to the site of treatment. Such chemokinesinclude, for example, RANTES, MCAF, MIP1-alpha, MIP1-Beta, IP-10 andcombinations thereof. The skilled artisan will recognize that certaincytokines are also known to have chemoattractant effects and could alsobe classified under the term chemokines. Other examples of molecules toinclude in the composition are immunogenic carrier proteins, such ashepatitis B surface antigen, keyhole limpet hemocyanin (KLH), bovineserum albumin (BSA), ovalbumin, mouse serum albumin or rabbit serumalbumin. Biological response modifiers (BRM) may be utilized that havebeen shown to upregulate T cell immunity or downregulate suppressor cellactivity. Such BRMs include, but are not limited to, cimetidine (CIM;1200 mg/d) (Smith/Kline, PA); low-dose cyclophosphamide (CYP; 300 mg/m²)(Johnson/Mead, NJ), or a gene encoding a protein involved in one or moreimmune helper functions, such as B-7.

In some aspects to the disclosure, an adjuvant in addition to thelipooligosaccharide/lipid A-based mimetic(s) is employed in animmunogenic composition. An example of an adjuvant is alum or squalene.

In one aspect, an adjuvant effect is achieved by use of an agent, suchas alum, used in about 0.05 to about 0.1% solution in phosphate bufferedsaline. Alternatively, the antigen is made as an admixture withsynthetic polymers of sugars (Carbopol®) used as an about 0.25%solution. Adjuvant effect may also be made my aggregation of the antigenin the composition by heat treatment. Some adjuvants, for example,certain organic molecules obtained from bacteria, act on the host ratherthan on the antigen. An example is muramyl dipeptide(N-acetylmuramyl-L-alanyl-D-isoglutamine [MDP]), a bacterialpeptidoglycan. The effects of MDP, as with most adjuvants, are not fullyunderstood. MDP stimulates macrophages but also appears to stimulate Bcells directly. The effects of adjuvants, therefore, are notantigen-specific. If they are administered together with a purifiedantigen, however, they can be used to selectively promote the responseto the antigen.

In certain embodiments, hemocyanins and hemoerythrins may also be usedin the invention. The use of hemocyanin from keyhole limpet (KLH) ispreferred in certain embodiments, although other molluscan and arthropodhemocyanins and hemoerythrins may be employed. Various polysaccharideadjuvants may also be used. Polyamine varieties of polysaccharides areuseful, such as chitin and chitosan, including deacylated chitin.Another group of adjuvants are the muramyl dipeptide (MDP,N-acetylmuramyl-L-alanyl-D-isoglutamine) group of bacterialpeptidoglycans. Derivatives of muramyl dipeptide, such as the amino acidderivative threonyl-MDP, and the fatty acid derivative MTPPE, are alsocontemplated.

Another adjuvant contemplated for use in the present invention is BCG.BCG (Bacillus Calmette-Guerin, an attenuated strain of Mycobacterium)and BCG-cell wall skeleton (CWS) may also be used as adjuvants in theinvention, with or without trehalose dimycolate. Trehalose dimycolatemay be used itself. Trehalose dimycolate administration has been shownto correlate with augmented resistance to influenza virus infection inmice (Azuma et al., 1988). Trehalose dimycolate may be prepared asdescribed in U.S. Pat. No. 4,579,945.

Amphipathic and surface active agents, e.g., saponin and derivativessuch as QS21 (Cambridge Biotech), form yet another group of adjuvantsfor use with the immunogens of the present invention. Nonionic blockcopolymer surfactants (Rabinovich et al., 1994; Hunter et al., 1991) mayalso be employed. Oligonucleotides are another useful group of adjuvants(Yamamoto et al., 1988). Quil A and lentinen are other adjuvants thatmay be used in certain embodiments of the present invention. One groupof adjuvants that are useful include detoxified endotoxins.

An antigenic composition of the present invention may be mixed with oneor more additional components (e.g., excipients, salts, etc.) that arepharmaceutically acceptable and compatible with at least one activeingredient (e.g., antigen). Suitable excipients are, for example, water,saline, dextrose, glycerol, ethanol and combinations thereof. Anantigenic composition of the present invention may be formulated as aneutral or salt form. A pharmaceutically-acceptable salt, includes theacid addition salts (formed with the free amino groups of the peptide)and those that are formed with inorganic acids such as, for example,hydrochloric or phosphoric acid, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. A salt formed with a freecarboxyl group also may be derived from an inorganic base such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxide, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and combinations thereof. In addition, ifdesired, an antigenic composition may comprise minor amounts of one ormore auxiliary substances such as for example wetting or emulsifyingagents, pH buffering agents, etc. which enhance the effectiveness of theantigenic composition or vaccine.

X. Vaccine and Immunogenic Composition Preparations

Once produced, synthesized and/or purified, an antigen or other vaccinecomponent may be prepared as a vaccine or immunogenic composition foradministration to an individual, wherein the vaccine or immunogeniccomposition comprises the lipooligosaccharide/lipid A-based mimetic. Thepreparation of a vaccine is generally well understood in the art, asexemplified by U.S. Pat. Nos. 4,608,251, 4,601,903, 4,599,231,4,599,230, and 4,596,792, all incorporated herein by reference. Suchmethods may be used to prepare a vaccine or immunogenic compositioncomprising an antigen as active ingredient(s) and alipooligosaccharide/lipid A-based mimetic(s), in light of the presentdisclosure. In particular embodiments, the compositions of the presentinvention are prepared to be pharmacologically acceptable.

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more lipooligosaccharide/lipid A-basedmimetics dissolved or dispersed in a pharmaceutically acceptablecarrier. The phrases “pharmaceutical or pharmacologically acceptable”refers to molecular entities and compositions that do not produce anadverse, allergic or other untoward reaction when administered to ananimal, such as, for example, a human, as appropriate. The means ofpreparation of a pharmaceutical composition that contains at least onelipooligosaccharide/lipid A-based mimetics or additional activeingredient will be known to those of skill in the art in light of thepresent disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference. Moreover, for animal (e.g., human) administration, it will beunderstood that preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, binders, excipients, disintegration agents, lubricants,sweetening agents, flavoring agents, dyes, such like materials andcombinations thereof, as would be known to one of ordinary skill in theart (see, for example, Remington's Pharmaceutical Sciences, 18th Ed.Mack Printing Company, 1990, pp. 1289-1329, incorporated herein byreference). The compositions may comprise different types of carriersdepending on whether it is to be administered in solid, liquid oraerosol form, and whether it need to be sterile for such routes ofadministration as injection. Except insofar as any conventional carrieris incompatible with the active ingredient, its use in the therapeuticor pharmaceutical compositions is contemplated.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

The composition may be formulated into a composition in a free base,neutral or salt form. Pharmaceutically acceptable salts, include theacid addition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays,aerosols or inhalants in the present invention. Such compositions aregenerally designed to be compatible with the target tissue type. In anon-limiting example, nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays.Nasal solutions are prepared so that they are similar in many respectsto nasal secretions, so that normal ciliary action is maintained. Thus,in preferred embodiments the aqueous nasal solutions usually areisotonic or slightly buffered to maintain a pH of about 5.5 to about6.5. In addition, antimicrobial preservatives, similar to those used inophthalmic preparations, drugs, or appropriate drug stabilizers, ifrequired, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

In certain embodiments the composition is prepared for administration bysuch routes as oral ingestion. In these embodiments, the solidcomposition may comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, or combinations thereof. Oralcompositions may be incorporated directly with the food of the diet.Preferred carriers for oral administration comprise inert diluents,assailable edible carriers or combinations thereof. In other aspects ofthe invention, the oral composition may be prepared as a syrup orelixir. A syrup or elixir, and may comprise, for example, at least oneactive agent, a sweetening agent, a preservative, a flavoring agent, adye, a preservative, or combinations thereof.

In certain preferred embodiments an oral composition may comprise one ormore binders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

XI. Vaccine and Immunogenic Composition Administration

The manner of administration of a vaccine or immunogenic composition maybe varied widely. Any of the conventional methods for administration ofa vaccine or immunogenic composition are applicable. For example, avaccine or immunogenic composition may be conventionally administeredintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intratumorally, intramuscularly, intraperitoneally,subcutaneously, intravesicularlly, mucosally, intrapericardially,orally, rectally, nasally, topically, in eye drops, locally, usingaerosol, injection, infusion, continuous infusion, localized perfusionbathing target cells directly, via a catheter, via a lavage, in creams,in lipid compositions (e.g., liposomes), or by other method or anycombination of the forgoing as would be known to one of ordinary skillin the art (see, for example, Remington's Pharmaceutical Sciences, 18thEd. Mack Printing Company, 1990, incorporated herein by reference).

An immunization schedule and dosages may be varied on a patient bypatient basis, taking into account, for example, factors such as theweight and age of the patient, the type of disease being treated, theseverity of the disease condition, previous or concurrent therapeuticinterventions, the manner of administration and the like, which can bereadily determined by one of ordinary skill in the art.

A vaccine or immunogenic composition is administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective and immunogenic. For example, theintramuscular route may be preferred in the case of toxins with shorthalf lives in vivo. The quantity to be administered depends on thesubject to be treated, including, e.g., the capacity of the individual'simmune system to synthesize antibodies, and the degree of protectiondesired. The dosage of the vaccine will depend on the route ofadministration and will vary according to the size of the host. Preciseamounts of an active ingredient required to be administered depend onthe judgment of the practitioner. In certain embodiments, pharmaceuticalcompositions may comprise, for example, at least about 0.1% of an activecompound. In other embodiments, the an active compound may comprisebetween about 2% to about 75% of the weight of the unit, or betweenabout 25% to about 60%, for example, and any range derivable thereinHowever, a suitable dosage range may be, for example, of the order ofseveral hundred micrograms active ingredient per vaccination. In othernon-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per vaccination, and any range derivabletherein. In non-limiting examples of a derivable range from the numberslisted herein, a range of about 5 mg/kg/body weight to about 100mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above. A suitable regime for initial administrationand booster administrations (e.g., innoculations) are also variable, butare typified by an initial administration followed by subsequentinoculation(s) or other administration(s).

In many instances, it will be desirable to have multiple administrationsof the vaccine or immunogenic composition, usually not exceeding siximmunizations, more usually not exceeding four immunizations and in somecases one or more, usually at least about three immunizations. Theimmunizations will normally be at from two to twelve week intervals,more usually from three to five week intervals. Periodic boosters atintervals of 1-5 years, usually three years, will be desirable tomaintain protective levels of the antibodies.

The course of the immunization may be followed by assays for antibodiesfor the supernatant antigens. The assays may be performed by labelingwith conventional labels, such as radionuclides, enzymes, fluorescents,and the like. These techniques are well known and may be found in a widevariety of patents, such as U.S. Pat. Nos. 3,791,932; 4,174,384 and3,949,064, as illustrative of these types of assays. Other immune assayscan be performed and assays of protection from challenge with theantigen can be performed, following immunization.

Following use of the lipooligosaccharide/lipid A-based mimetic in animmunogenic composition in an individual, an immune response isenhanced. The enhanced immune response may be an active or a passiveimmune response.

XII. Kits

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, one or more lipooligosaccharide/lipid A-basedmimetics and/or bacterial strains to produce them and/or reagents formodifying the bacteria are comprised in a kit. In certain aspects, oneor more reagents for modifying, culturing, and/or extracting frombacteria are include in the kit.

In specific embodiments, one or more lipooligosaccharide/lipid A-basedmimetics are included in the kit and may or may not be formulated withanother agent. The other agent may be an immunogenic composition itself,such as a vaccine, or it may be part of an immunogenic composition, suchas an antibody, a weakened microbe, a killed microbe, one or moreantigens, a toxoid, polysaccharide, or nucleic acid. Thelipooligosaccharide/lipid A-based mimetics may be formulated fordelivery to a mammal or may be provided with one or more reagents toproduce a formulation for delivery to a mammal. The bacterial strain ofthe kit may be provided as a bacterial stab, bacterial slant, frozenglycerol stock, or freeze dried powder, as examples. The bacteria may beprovided in the kit at a particular desired temperature, includingbetween frozen (−80° C.) and room temperature. In embodiments, the kitcomprises one or more reagents for modifying a bacteria, such asreagents to handle a recombinant vector and/or reagents to assay thebacteria for presence of the vector (such as PCR reagents, restrictionenzymes, polymerases, ligases, buffers, nucleotides, etc.).

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquotted. Where there are more than one component in the kit, the kitalso will generally contain a second, third or other additionalcontainer into which the additional components may be separately placed.However, various combinations of components may be comprised in a vial.The kits of the present invention also will typically include a meansfor containing the compositions in close confinement for commercialsale. Such containers may include injection or blow-molded plasticcontainers into which the desired vials are retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. In some cases, thecontainer means may itself be a syringe, pipette, and/or other such likeapparatus, from which the formulation may be applied to a desired areaof the body, injected into an animal, and/or even applied to and/ormixed with the other components of the kit. However, the components ofthe kit may be provided as dried powder(s). When reagents and/orcomponents are provided as a dry powder, the powder can be reconstitutedby the addition of a suitable solvent. It is envisioned that the solventmay also be provided in another container means. The kits may alsocomprise a container means for containing a sterile, pharmaceuticallyacceptable buffer and/or other diluent.

XIII. Exemplary Bacterial Strains

Embodiments of the disclosure employ Gram negative bacterial strains toproduce a desired composition. The Gram negative bacteria may be of anykind, including from Acetobacter, Borrelia, Bortadella, Burkholderia,Campylobacter, Chlamydia, Enterobacter, Eshcerichia, Fusobacterium,Helicobacter, Hemophilus, Klebsiella, Legionella, Leptospiria,Neisseria, Nitrobacter, Proteus, Pseudomonas, Ricketsia, Salmonella,Serratia, Shigella, Thiobacter, Treponema, Vibrio, or Yersinia. Inspecific embodiments, one or more of the following bacteria are utilizedin methods of the disclosure: Acetic acid bacteria, Acinetobacterbaumannii, Agrobacterium tumefaciens, Anaerobiospirillum, Arcobacter,Arcobacter skirrowii, Armatimonas rosea, Bacteroides, Bacteroidesfragilis, Bacteroides ruber, Bartonella taylorii, Bdellovibrio,Brachyspira, Cardiobacterium hominis, Chthonomonas calidirosea, Coxiellaburnetii, Cyanobacteria, Cytophaga, Dialister, Enterobacter,Enterobacter cloacae, Enterobacter cowanii, Enterobacteriaceae,Enterobacteriales, Escherichia, Escherichia coli, Escherichiafergusonii, Fimbriimonas ginsengisoli, Fusobacterium necrophorum,Fusobacterium nucleatum, Fusobacterium polymorphum, Haemophilushaemolyticus, Haemophilus influenzae, Helicobacter, Helicobacter pylor,Klebsiella pneumoniae, Legionella, Legionella pneumophila, Leptotrichiabuccalis, Escherichia coli, Luteimonas aestuarii, Luteimonas aquatica,Luteimonas composti, Luteimonas lutimaris, Luteimonas marina, Luteimonasmephitis, Luteimonas vadosa, Megamonas, Megasphaera, Meiothermus,Methylobacterium fujisawaense, Morax-Axenfeld diplobacilli, Moraxella,Moraxella bovis, Moraxella osloensis, Morganella morganii,Negativicutes, Neisseria cinerea, Neisseria gonorrhoeae, Neisseriameningitidis, Neisseria sicca, Nitrosomonas eutropha, Nitrosomonashalophila, Nitrosomonas oligotropha, Pectinatus, Pelosinus, Pontiacfever, Propionispora, Proteobacteria, Proteus mirabilis, Proteuspenneri, Pseudomonas, Pseudomonas aeruginosa, Pseudomonas, Pseudomonasluteola, Pseudoxanthomonas broegbernensis, Pseudoxanthomonas japonensis,Rickettsia rickettsii, Salmonella, Salmonella bongori, Salmonellaenterica, Salmonella enterica subsp. enterica, Selenomonadales, Serratiamarcescens, Shigella, Sorangium cellulosum, Sphaerotilus, Spirochaeta,Spirochaetaceae, Sporomusa, Stenotrophomonas, Stenotrophomonasnitritireducens, Thermotoga neapolitana, Trimeric autotransporteradhesin, Vampirococcus, Verminephrobacter, Vibrio adaptatus, Vibrioazasii, Vibrio campbellii, Vibrio cholerae, Vitreoscilla, Wolbachia, orZymophilus.

The bacterial strain to be utilized ideally is an avirulent strain. Theskilled artisan recognizes that any naturally virulent bacteria may begenetically engineered to be avirulent or otherwise rendered to beavirulent by any means, including loss of one or more virulence factors,such as loss of at least one virulence plasmid and/or bacteriophage. Insome cases, a naturally occurring avirulent version of a normallyvirulent bacteria may be employed.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 General Embodiments

In particular embodiments, one can generate a variety of modifiedlipooligosaccharide (LOS), consisting of core regions of LPS and lipid Amolecules created by heterologous expression from plasmids or thechromosome of single or combinations of acyltransferase, deacylases,phosphatase and/or glycosyltransferases enzymes or through mutation ofglobal regulatory genes required for regulating lipid A biosynthesis inan attenuated (BSL2-safe) hypo-acylated Y. pestis background. Thisexemplary strain when grown at mammalian temperatures produces abisphosphorylated tetra-acylated structure that does not elicitproinflammatory responses in a variety of in vitro and in vivo assays.Methods of the disclosure allow one to use enzymes obtained from a widevariety of bacterial backgrounds (Table 1). These enzymes havespecificities for fatty acid chain length additions either on thediglucosamine backbone of lipid A or acyl-oxy-acyl on these primaryfatty acids, the removal of specific fatty acids or phosphate residuesfrom the diglucosamine backbone, or more global changes due to directedmutations in either the sensor kinase or response regulator of varioustwo-component regulatory systems, for example. Individually modified,rationally-designed lipid A structures are characterized using a varietyof mass spectrometry and gas chromatography-based methodologies (asexamples) to confirm the overall structure of the lipid A in thedisclosed strains to generate using BECC.

Modified LOS/Lipid A structures can be tested in both in vitro and inhighly relevant in vivo animal model(s) as stand-alone immunotherapeuticmolecules or adjuvants in new immunogenic (including vaccine)formulations. As compared to impure and low-output MPL or AGPs,respectively, engineered lipid A-based structures can be: (1) producedefficiently, (2) fully characterized using mass spectrometry and gaschromatography-based methodologies for structure and purity, and (3)produced rapidly in sufficient quantities for use in preclinical andclinical testing and for clinical use. Additionally, adjuvanticity witha subset of LOS/lipid A structures are confirmed using an engineeredmurine strain expressing human TLR4 (humanized TLR4; Hajjar et al.,2012) to more closely mimic human immune responses and address potentialdiscordance in LPS recognition by human compared with mouse TLR4/MD-2(Kawai and Akira, 2011; Pasare and Medzhitov, 2005). Ultimately,assessment of the adjuvant characteristics of BECC synthesized moleculesvia murine and human TLR4 results in an increased understanding ofhumoral and cell-mediated immunity to a variety of ligands that induceT_(H)1-T_(H)17- and T_(H)2-type immune responses in the systemic andmucosal compartments of the host, in at least some embodiments.

Once individual structures are verified, one can test theimmunotherapeutic potential of these new molecules using standard,proven in vitro and in vivo assay to determine if any of these moleculesrepresent adjuvants and/or immunomodulating reagents. One can alsoinclude a well-known available reference adjuvant, such as MPL as ahead-to-head comparison to the molecules synthesize by BECC. Embodimentsof the disclosure provide major implications in the field of antigenrecognition, formulation, and vaccine design.

Example 2 Construction of Rationally-Designed Los/Lipid a Structureswith Engineered Lipid a Modifications Using Bacterial EnzymaticCombinatorial Chemistry (BECC)

In one aspect of the disclosure, there is custom synthesis of LOS/lipidA molecules using heterologous expression of bacterial enzymes requiredfor LPS biosynthesis (acyltransferases, glycosyltransferases,phosphatases, and/or kinases, for example), and through manipulation ofglobal regulatory or biosynthesis genes in an attenuated Yp background(as an example of a background). A variety of mass spectrometry and gaschromatography-based methodologies can be used to verify that thecorrect lipid A modifications have been synthesized.

In initial analysis, there is synthesis of proof-of-conceptadjuvant/immunotherapeutic LOSs with modified lipid A anchors. Inspecific embodiments, three BECC Yp strains that produce uniqueLOS/lipid A structures when grown at 37° C., mammalian host temperature(Table 1) are provided herein. BECC-generated LOS were tested in invitro stimulation studies; the ΔphoP 37° C. tetra-acylated structure wascompared directly to the approved adjuvant, MPL, in an in vivo,vaccination study described elsewhere herein.

Construction of additional rationally-designed LOS/lipid A structureswith modified lipid A structures using BECC is achieved. One can createand validate safe-to-use, attenuated Y. pestis (Yp) strains withmodified LOS/lipid A molecules that are subsequently screened forimmunogenic potential in vitro and in vivo. Useful to the design of BECCare well-characterized enzymes sourced from a variety of bacterialspecies (FIG. 8 provides a list of cloned enzymes). Novel to BECC is theheterologous expression of these modifying enzymes in an attenuated Ypbackground, such as KIM6 or KIM10, to engineer unique lipid Astructures. Bacterial conjugation, electroporation, or bacterial matingmay be utilized to introduce genes from an enzyme library into the YpKIM6 parent chromosome or via exchange of expression plasmids (Jones etal., 2010; Rebeil et al., 2004; Donenberg and Kaper, 1991). An exampleof a scheme of use for the individual modifying genes is based on knownstructure/function interaction of specific modifications and the hostinnate immune receptor (TLR4) and may be prioritized as follows: 1)mutations in global regulators of lipid A modifications, 2) altering thelevel of fatty acid content through the addition or deletion ofacyltransferases and/or deacylases, 3) expression of phosphatase tospecifically remove one of the terminal phosphates on the lipid Abackbone, and 4) expression of glycosyltransferases that will addspecific sugar residues to the terminal phosphate residues. Strains maybe vetted by a combination of MALDI-TOF MS for initial lipid Astructural confirmation followed by higher order MS (ESI) to provideintimate structural detail and molecular design validation. One can usegas chromatography (GC) to quantify the percentage of total fatty acidsof each respective modified lipid A structure, providing furtherverification that the correct strains have been generated and that theyare both productive and accurate.

TABLE 1 BECC MODIFIED STRAINS OF YP Strains Strain Description 1.Wild-type Yp WT KIM6 (exempt select agent strain of Yp; lacks KIM6pigmentation locus and pCD1 plasmid; CDC BSL-2 classification) 2.KIM6-pagP+ KIM6 with a repaired pagP gene (adds C16 fatty acid to lipidA) 3. ΔphoP-KIM6 KIM6 with a deleted phoP gene, which is a member of a 2component sensor kinase signaling system (a transcriptional regulator)4. ΔphoP -pagP+ KIM6 with a deleted phoP gene which is a member of a 2component sensor kinase signaling system (a transcriptional regulator)and a repaired pagP activating gene which adds C16 fatty acids to lipidA

Modifications in these strains have been confirmed by MS and GC analysis(data not shown). Corresponding major lipid A structures are indicatedin FIG. 1.

Analysis of LOS and lipid A isolated from BECC constructed strains isprovided herein. For initial screening, one can use two small-scale LOSextraction protocols that require small overnight cultures (˜5 mls).These methods include a phenol-based (Yi and Hackett, 2000; Westphal andJuan, 1965) and an ammonium hydroxide/isobutyric acid-based (El Hamidiet al., 2005) protocol, which are repeatable and robust extractiontechniques, standard methods in the Ernst laboratory. After extraction,lipid A will be liberated from these LOS preparations using gentlehydrolysis, which preserves structural elements (e.g., phosphate groupsand attached carbohydrate moieties) that are sensitive to harsh acidtreatment (Caroff et al., 1988) One can use a variety of massspectrometric-based techniques, such as MALDI-TOF and ESI routinely usedin the art to characterize the base structure of the lipid A in both thenegative and positive-ion mode (Ernst et al., 2006). Large-scale LOSpreparations are extracted using a hot phenol/water extraction method(Ernst et al., 2007; Ernst et al., 2006; West et al., 1997; Hajjar etal., 2006). Subsequently, LOS are treated to ensure purity fromcontaminating nucleic acids and proteins (Fischer et al., 1983) andconverted to lipid A by mild hydrolysis. LOS samples are extracted toremove contaminating phospholipids (Folch et al., 1957) and TLR2-agonistproteins (Hirschfeld et al., 2000) thus generating preparations suitablefor structural analysis and proinflammatory studies discussed proposedbelow. LOS/lipid A fatty acid content is measured by gas chromatography(GC) after acid hydrolysis, methylation, and hexane extraction (Guo etal., 1997; Somerville et al., 1996). The resultant MS^(n) and GC data isused to define the exact structure of individual molecules present inthe isolated lipid A from the WT and BECC constructed strain.

Example 3 Define Immunomodulatory Profile of Engineered Los/Lipid aUsing an In Vitro Screening Model

In this embodiment, it is demonstrated that the rationally-designed,validated molecules generated by methods herein produce altered innateresponses. One can characterize BECC synthesized molecules for innateimmune responses mediated via TLR4 in a dose dependent manner to allowfor the selection of candidate molecules for use in in vivoadjuvanticity-based assays. One can utilize murine macrophage cell lines(RAW and MH-S) and human cell lines (U937 and THP-1) along with primarymacrophage cells from mice (PES) and humans (PBMCs obtained from Lonzaas buffy coats) in cell stimulation studies using a wide dose range ofLOS/lipid A. Both LOS and lipid A preparation are tested to rule out anyrole for the core region of LOS in altering innate immune responses.Preliminary cell stimulation screening of supernatants is performedusing cytokine/chemokine ELISAs (Hajjar et al., 2006; Darveau et al.,1995; Guo et al., 1998; Grkovich et al., 2006). Stimulation of TLR4 andbroad innate immune activation via the NF-κB and TRIF/TRAM arms ofproinflammatory signaling pathways will be demonstrated by secretion ofIL-8 and RANTES/IL-1β. TLR4 signaling through NF-κB (MyD88-dependent)results in secretion of the neutrophil recruiting chemokine IL-8,similarly TRIF/TRAM (MyD88-independent) will also promotelymphoattraction, but through the expression of RANTES (CCL5) driven bythe transcription factor AP-1. These chemokines contribute to prompt,effective immune activation and help describe the mechanism-of-actionfor LOS-based adjuvant molecules. Deep profiling of cell stimulation bymodified LOS/lipid A is performed using cytokine multiplex assays toillustrate activation of cell-mediated and/or humoral pathways (Coler etal., 2011; Man et al., 2010; West et al., 2008). To supplement thesecreted protein profiling, RNA harvested from stimulated human cells isanalyzed by qPCR for cytokine/chemokine gene expression (Meng et al.,2011; Coats et al., 2009). Selection of candidate molecules for use inthe subsequent in vivo screening assays is based on combination andrank-ordering of the cytokine and gene expression profiles. Priority canbe given to molecules that have low toxicity and remainimmunostimulatory as defined by the cytokine profile (i.e.: triggeringchemotaxis, but not profound apoptosis or pyroptosis). Particularmodified LOS/lipid A molecules are forwarded into in vivo screening inat least some cases. This strategy delineates a rational triage scheduleto screen potential immunotherapeutic candidates.

Highlighting the feasibility of using an in vitro screening model toprogress candidate molecules to further in vivo work is illustrated inFIG. 2. FIG. 2 demonstrates that an engineered LOS with hexa-acylatedlipid A from the KIM6 pagP expressing strain grown at mammalian hosttemperature induces secreted cytokines IL-8 and RANTES in contrast tothe absence thereof by the WT base. The pagP enzyme is non-functional inall Yp strains. MPL (from major manufacturers) tested over the samedose-response manner only respond at the very highest doses. A 4-logdose increase was necessary to elicit a RANTES response approaching theengineered LOS at 0.1 ng/mL (pagP+37° C.). Furthermore, ˜3-folddifferences in response were observed between the two sources of MPLeven at 1 μg/ml. Therefore, structural heterogeneity of MPL resultantfrom the nature of chemical synthesis (FIG. 1) alters its potencydramatically. These data illustrate engagement of both theMyD88-dependent and -independent arms of the TLR4 signaling pathway bymodified LOS. Surprisingly, LOS from the pagP expressing, ΔphoP strainis a poor inducer of either cytokine readouts, suggesting an importantrole for this global regulator in controlling more global Yp lipid Amodifications. GC analysis shows that the ΔphoP-pagP+ strain has ˜50%less C16 fatty acid in its lipid A as compared to the pagP+ lipid A.

Example 4 Define Efficacy of Immunomodulatory Candidates as Adjuvantsand/or Stand-Alone Agents Using In Vivo Murine Screening Models

In one embodiment of the disclosure, the immunogenic potential of acandidate molecule to alter innate and adaptive responses in vivo isdemonstrated. One can develop molecules that will have low cytoxicityyet retain the ability to activate the innate and adaptive immuneresponses in vivo and also confer protection against virulent bacterialstrains in a murine vaccine model as highlighted elsewhere herein.Particular molecules generated herein that are potent immune stimulantswithout being overtly cytotoxic are administered to mice both alone andwith known immunogenic reagents to probe innate and adaptive immunepotentiation, specifically direct modulation of cell mediated andhumoral immunity and balancing of Th₁/Th₂ response. Additionally, theuse for LOS/lipid As as an immunoprophylactic to a lethal challenge ofinfectious agent is analyzed. In at least some cases, there are miceexpressing murine TLR4 alone and with known immunogens to accesstoxicity and activation of the innate and adaptive immune responses inmurine vaccine models (intranasal and intramuscular). Subsequently,LOS/lipid A structures with potential adjuvanticity are confirmed usingan engineered murine strain expressing human TLR4 to more closely mimichuman immune responses.

Modified LOS from strains in the WT Yp background can induce an adaptiveimmune response when used with other known immunogenic reagents in invivo murine vaccine experiments. The ability to design and useGram-negative bacterial strains containing alterations in lipid Astructure that confer protection in in vivo murine disease models(Hajjar et al., 2006; Lai et al., 2010; Kanistanon et al., 2008; West etal., 2008; Lembo et al., 2008) is known, and validation of this strategyis shown in FIG. 3. These data show proof-of-principle that modified LOSused as an adjuvant in combination with Yp capsular F1 Ag evokes anadaptive immune response equal to the approved adjuvant MPL; inducing aIgG2c antibody response, indicative of a Th₁ response, as well as aTh₂-driven IgG1 response equal to MPL and ODN2395 (CpG) in a murinemouse model. This mode of adaptive immune activation is TLR4-dependentas it is not observed in TLR4-KO mice and is suggestive of a balancedTh₁/Th₂ profile that is an asset for optimization of immunogeniccomposition design, including vaccine design.

One can define cytotoxicity of engineered molecules using in vivo murinescreening models. To determine the toxicity in eachbiologically-relevant candidate, escalating doses (0.5 μg, 5 μg, 50 μg,with mock controls) are delivered by intraperitoneally (IP) injection toC57BL/6 (The Jackson Laboratory), 5 mice/dose group. Mice are followedfor survival and signs of acute toxicity. Necropsies are performed atstudy-end to survey signs of damage and/or toxicity by gross pathologyor histology (as required). Compounds with absent or acceptably lowtoxicity are carried forward for further characterization.

One can demonstrate direct activation of the innate immune system byengineered LOS. To test the ability of modified LOS to stimulate theinnate immune system in vivo, candidate escalating doses (0.5 μg, 5 μg,50 μg with antigen only, MPL, and mock controls, as examples), areinjected by the IV route into C57/BL6 mice. Sera are collected 2 hourspost-injection for analysis. Cytokine profiles are determined bymultiplex assays or ELISA and analyzed for specific early activationmarkers (ex: TNF-α, IL-1β, III III.).

An immunization strategy using LOS to potentiate adaptive immuneresponses in vivo is included in the disclosure.

To demonstrate that candidate molecules will activate adaptive immuneresponses in an in vivo murine model, C57/BL6 mice are randomly dividedinto groups, each consisting of five mice. Animals are dosedintraperitoneally (IP) with 25 μg of Yp F1 antigen alone and with 50 μgof candidate LOS, or MPL/ODN2395 (CpG) as controls (with necessary mockcontrols). 50 μg of the candidate LOS are also administered alone totest for stand-alone immunotherapeutic efficacy. Boosting injection isgiven 4 weeks later. Sera is collected 1 week after the secondimmunization to quantitate F1 antigen-specific antibodies (total IgGinitially, class and subtype as relevant) by ELISA; concentrations ofserum cytokines, hallmarks of immune activation (IL-2, IL-4, IFN-γ,TNF-α, IL-12, IL-8, etc.), will also be profiled by cytokine multiplexassays.

One can characterize protective immune responses to virulent Y. pestisconferred by LOS-based vaccine. Mice previously vaccinated (above) withcandidate LOS adjuvant formulations will be used to test the durabilityof the induced adaptive immune responses from a lethal challenge usingthe fully virulent CO92 strain of Yp. Two weeks after the second boostdose (study week 6), mice will be challenged with ˜100×LD₅₀ (200-300cfu) of CO92 by IP injection. Mice will be monitored for survival andsigns of disease for 4 weeks.

In specific embodiments, any antigen is employed with one or morecompositions of the disclosure.

Example 5 Generation of Rationally-Designed Adjuvants have MajorImplications in the Field of Antigen and Adjuvant Formulation, andVaccine Design

Synthesis of adjuvant/immunotherapeutic LOS with modified lipid Astructures is performed. BECC Yersinia pestis (Yp) strains are generatedthat produce unique lipooligosaccharide (LOS)/lipid A structures whengrown at host (37° C.) or environmental (26° C.) temperatures. Plasmidsexpressing individual lipid A modifying enzymes were expressed in transin the genetically tractable, avirulent Yp strains KIM6 or KIM10. Bothstrain backgrounds are exempt from select agents status and are approvedfor use under BSL-2 laboratory practices as they lack the pigmentationlocus (pgm) and the pCD1 plasmid required for virulence(http://www.selectagents.gov). Examples of two of the BECC designedmolecules, as compared to the structure of an AGP and MPL, are shown inFIGS. 4A-4E. To date, these BECC-generated LOS have been tested ininitial in vitro stimulation studies; the ΔphoP 37° C. tetra-acylatedstructure has performed similarly to the approved adjuvant, MPL in an invivo, proof-of-concept vaccination study below.

BECC-synthesized LOS molecules have altered innate immune responses.Highlighting the feasibility of using an in vitro screening model toprogress candidate molecules to further in vivo work is illustrated inFIG. 5. FIG. 5 demonstrates that an engineered LOS with hexa-acylatedlipid A from the KIM6 pagP expressing strain grown at mammalian hosttemperature stimulated secretion of cytokines IL-8, RANTES, and IL-23from THP-1 cells. MPL (GSK and Avanti) tested over the samedose-response manner only respond at the very highest doses. A 4-logdose increase was necessary to elicit a RANTES response approaching theengineered LOS at 0.1 ng/mL (pagP+37° C.). Furthermore, ˜3-folddifferences in response were observed between the two sources of MPLeven at 1 μg/ml. Therefore, MPL potency suffers dramatically fromstructural heterogeneity, a result of the nature of chemical synthesis(FIGS. 4A-4E). These data illustrate that modified LOS engages both theMyD88-dependent and -independent arms of the TLR4 signaling pathway(FIG. 7). Surprisingly, LOS from the pagP expressing, ΔphoP strain is apoor inducer of either cytokine readouts, suggesting an important rolefor this global regulator in controlling more global Yp lipid Amodifications. GC analysis shows that the ΔphoP-pagP+ strain has ˜50%less C16 fatty acid in its lipid A, as compared to the pagP+ lipid A.

Modified LOS enhances adaptive immune response to protein antigen. It isdemonstrated that modified LOS from strains in the WT Yp background caninduce an adaptive immune response when used with other knownimmunogenic reagents in in vivo murine vaccine experiments. Validationof this strategy is shown in FIG. 6. These data show thatBECC-synthesized Yp LOS can be used as an adjuvant in combination withYp capsular F1-V Ag and evokes an adaptive immune response equal to theapproved adjuvant MPL and ODN2395 (CpG) in a murine mouse model. Thismode of adaptive immune activation is TLR4-dependent as it is notobserved in TLR4-KO mice and indicates that BECC-synthesized adjuvantsare an asset for optimization of vaccine design.

Example 6 Construction of Rationally-Designed Los/Lipid a Structureswith Engineered Lipid a Modifications Using Bacterial EnzymaticCombinatorial Chemistry (BECC)

An area of great interest is in the development of rationally-designedadjuvants based on molecules that stimulate the host innate immunesystem, specifically pattern-recognition receptors, including toll-likereceptors (TLR). The objective of this aim is to custom synthesizeLOS/lipid A molecules using heterologous expression of acyltransferases,glycosyltransferases, phosphatases, kinases, and modification of globalregulatory genes required for lipid A biosynthesis with altered hostinnate immune responses.

Construction of additional rationally-designed LOS/lipid A structureswith modified lipid A structures using bacterial enzymatic combinatorialchemistry (BECC) is encompassed in the disclosure. One can engineermodified LOS/lipid A molecules in an Yp background for determination ofadjuvant potential using in vitro and in vivo assay systems. Briefly,one can heterologously express lipid A modifying enzymes in transthrough bacterial conjugation or electroporation in a safe, attenuatedBSL-2 Yp background (KIM6 or KIM10) to engineer unique lipid Astructures (Donnenberg and Kaper, 1991; Rebeil et al., 2004; Jones etal., 2010) These Yp strain backgrounds were chosen as they produce abisphosphorylated tetra-acylated lipid A structure (also known aslipidIVA) when grown at 37° C., thus allowing easy analysis of anyresulting modification(s). As shown above, specific enzymes (PagP andMsbB) have been expressed in these Yp strain backgrounds to generateunique proof-of-concept lipid A structures. In addition, there areengineered strains that lack the two-component transcriptional regulator(PhoP) required for global lipid A modifications. These lipid Amolecules are currently being evaluated for altered innate immuneresponses and adjuvant potential. These molecules are capable ofinducing immune responses that can lead to either TH1 and/or TH17responses. T helper responses are essential to the development of highaffinity and long-term protective immunity. The specific T helperresponse can alter antibody isotype that can effect affinity, andavidity, as well as interactions with other immune cells required forpathogen clearance. To date, all previously reported BECC-derivedmolecules are bisphosphorylated (phosphate moieties at the 1 and 4′position). As has been shown for the AGP and MPL molecules, the presenceof the phosphate moiety at the 1 position on the glucosamine backbone oflipid A plays an important role in host innate immune recognition.Therefore, one can focus on expressing enzymes that remove the terminalphosphate moieties in the strains that already have been generated byBECC. Two phosphatases have been identified in Francisella novicida thatremove the 1 position (LpxE) and 4′ position (LpxF) phosphatases (Wanget al., 2004; Wang et al., 2007) One can generate six additionalbacterial mutants that express acyltransferases (LpxP, HtrB, LpxXL) andglycosyltransferases (addition of aminoarabinose and galactosamine) ordeletions in other global regulators (PmrA/B) using plasmid constructscurrently available in an enzyme expression library (Raetz et al.,2007).

Aspects of the disclosure provide analytical validation of LOS and lipidA isolated from BECC constructed strains. For initial screening, one canuse two small-scale LOS extraction protocols that require smallovernight cultures (˜5 mls). These methods include a phenol-based (Yiand Hackett, 2000; Westphal and Juan, 1965) and an ammoniumhydroxide/isobutyric acid-based (El Hamidi et al., 2005) protocol, whichare repeatable and robust extraction techniques and standard methods inthe Ernst laboratory. After extraction, lipid A is liberated from theseLOS preparations using gentle hydrolysis, which preserves structuralelements (e.g., phosphate groups and attached carbohydrate moieties)that are sensitive to harsh acid treatment (Caroff et al., 1988). Onecan use a variety of established mass spectrometric-based techniques,such as MALDI-TOF and ESI, to characterize the base structure of thelipid A in both the negative- and positiveion mode (Ernst et al., 2006).Large-scale LOS preparations are extracted using a hot phenol/waterextraction method (Hajjar et al., 2006; Ernst et al., 2006; West et al.,1997; Ernst et al., 2007).

Subsequently, LOS are treated to ensure purity from contaminatingnucleic acids and proteins (Fischer et al., 1983), extracted to removecontaminating phospholipids (Folch et al., 1957) and TLR2-agonistproteins (Hirschfeld et al., 2000), and when required, converted tolipid A by mild hydrolysis. These steps ensure the generation ofpreparations suitable for structural analysis and proinflammatory andadjuvanticity experiments proposed below. LOS/lipid A fatty acid contentare measured by gas chromatography (GC) after acid hydrolysis,methylation, and hexane extraction (Guo et al., 1997; Somerville et al.,1996). The resultant MS and GC data is used to define the exactstructure of individual molecules present in the isolated lipid A fromthe WT and BECC constructed strain.

Example 7 Immune Responses to Novel Los/Lipid a Molecules

BECC-synthesized molecules produce innate immune responses mediated viaTLR4 in a dose-dependent manner, similar to AGP and MPL and theresulting analysis of host immune responses allow for the selection ofcandidate molecules for use in in vivo adjuvanticity studies.

Current lipid A mimetics, such as AGP and MPL differentially activatethe NF-KB and TRIF/TRAM signaling pathways, as compared to LPS leadingto altered secretion profiles of proinflammatory cytokines andchemokines. One can demonstrate that the rationally-designed, validatedmolecules produce altered innate responses. Initially, one can utilizeHEK293 cells transfected with TLR4 to determine host innate immuneresponses to our BECC synthesized molecules (Rebeil et al., 2004;MacArthur et al., 2011; Hajjar et al., 2006; Miller et al., 2005).Subsequently, as many adjuvants induce the innate immune response bypromoting dendritic cell (DC) cell maturation resulting in the primingof naive T cells and initiating potent immune responses, one can testLOS/lipid A preparations in murine and human DCs (Arias et al., 2012;Seregin et al., 2011). Both LOS and lipid A preparations are tested torule out any role for the core region of LOS in altering innate immuneresponses.

TLR4 restriction/dependence of novel engineered LOS/lipid A molecules isdemonstrated. To determine the restriction of the molecules to TLR4,HEK293 cells are utilized. HEK293 cells are transfected with murine orhuman MD2/TLR4 and incubated with increasing concentrations (0.01 ng/ml.0.1 ng, ml, 1 ng/ml, 10 ng/ml, 100 ng/ml) of novel LOS and lipid Apreparations for 4 and 24 hours. Supernatant is harvested and analyzedfor cytokine production (TNF-α, IL-6, IL-12 p40, IL-23 p19, RANTES) byELISA. Analysis of these cytokines can confirm that the BECC synthesizedmolecules are only active when MD-2/TLR4 is expressed and determinewhether the molecules are signaling through MyD88-TIRAP, TRIF-TRAM, or acombination of both. As can be seen in FIG. 7, the TLR4 signalingadapter can influence the T helper cell response to either a TH1 aloneor a combination of TH1 and TH17. T helper subtype have a marked effecton the subtype of antibody produced, which in turn can help or hinderthe effectiveness of the antibody response. Finally, to determinecellular cytotoxicity, the release of LDH in the supernatant will bemeasured by colorimetric assay.

There is maturation of Dendritic Cells upon novel adjuvant treatment.Dendritic cells (DCs), the quintessential antigen presenting cells(APC), have the ability to interact with many immune partnerssimultaneously. To determine if DCs are activated by theBECC-synthesized molecules, one can stimulate both bone marrow derivedDCs from mice, as well as peripheral blood DCs isolated from humandonors as described above. Supernatants are harvested and analyzed forcytokine production (the interferon-γ induced chemoattractants CXCL10and CXCL11, the T cell growth factor IL-2, the inflammatory mediatorsRANTES, and IL-6, as well as the growth factor GM-CSF) by ELISA ormultiplex cytokine arrays. In addition to secreted cytokines levelanalysis, cells are harvested and their maturation is quantified bymulticolor flow cytometry for the co-stimulatory molecules CD80 (B7-1)and CD86 (B7-2), as well as CD40 and MHC-class II. Increased surfaceexpression of these molecules is a marker for the ability of DCs topresent antigen and stimulate adaptive immune cells (Seregin et al.,2011; Coler et al., 2011).

Priority can be given to molecules that have, in the following order:(1) low toxicity, (2) remain immunostimulatory as defined by thecytokine profile (i.e., minimal NFKB activity; elevated TRW/TRAM andTH17 activity) and/or (3) have shown positive adjuvanticity potential inpreliminary experiments (Yp ΔphoP mutant).

Example 8 Determine Immune Responses for Adjuvant Candidates Using InVivo Murine Screening Models

In particular embodiments, a low cellular cytotoxicity TLR4 mimetic hasthe ability to active the innate and adaptive immune responses in vivoand to confer protection against lethal bacterial infection in murinevaccine models. Two particular molecules (a T_(H)1 and T_(H)1/T_(H)17modulator) identified by methods of the disclosure as beingdiscriminating immune stimulants without overt cytotoxic effects areadministered to mice both alone and with known immunogenic reagents toprobe innate and adaptive immune potentiation, specifically the directmodulation of either a strict T_(H)1 or a mixed immune responseT_(H)1/T_(H)17.

There is determination of cytotoxicity of and cytokine response toBECC-synthesized molecules using in vivo murine models. To determine thepotential toxicity of each candidate molecule, serum sampling, as wellas organ histology is carried out as described in Scheme 1. Serumsamples are collected from all mice prior to any manipulation to measurebaseline enzyme and cytokine levels. Two week after this initial serumharvest, escalating doses of BECC synthesized LOS or lipid A (0.5 μg, 5μg, 50 μg, with mock control) are delivered by the intranasal (IN) routeto C57BL/6 mice (n=3, The Jackson Laboratory). In an exemplary scheme(Scheme 1) for inoculation and sampling schedule to determine LOS/lipidA toxicity, on Day 0 a baseline serum is obtained, followed by adjuvantdose #1 at Day 14, serum harvest at day 28, adjuvant does #2 and serumharvest at day 42, serum harvest at day 56, and organ and serum harvestat day 60.

Both LOS and lipid A preparation are tested to determine if the coreregion of LOS plays a role in potential adjuvant responses. The IM routemodels the classical route of vaccine injection, while the IN routeshould induce mucosal-targeted immune responses. Mice are followed dailyfor survival and signs of acute toxicity. Every two weeks, peripheralblood is drawn for serum analysis. Analysis for toxicity in serumincludes measurements for kidney (creatinine, BUN) and liver (ALT, AST,INR and total protein) damage by either ELISA or enzymatic assay. At onemonth after the initial dosing, mice receive a second dose of adjuvantand followed for an additional month. Necropsies are performed atstudy-end to survey signs of damage and/or toxicity by gross pathologyor histology. Serum samples are also profiled by cytokine multiplexassays for the production immune activation (IL-2, IL-4, IFN-γ, TNF-α,IL-12 p40 or p70, IL-8). BECC synthesized molecules that have little tono toxicity, as well as robust cytokine profiles are carried forward forfurther characterization.

Evaluation of antigen-specific responses driven by engineered adjuvantsis performed. To evaluate the ability of the BECC-synthesized moleculesto generate antigen specific responses, two different bacterial antigensare assayed, F1-V capsular antigen (F1-V) from Yersinia pestis, as wellas Pertussis Toxin (PT) from Bordetella pertussis. The F1-V antigen hasbeen shown to be immunogenic and impart a protective immune responsewhen combined with a known adjuvant (Heath et al., 1998; Jones et al.,2006). The PT antigen was chosen to demonstrate the ability of thehelper cell polarization, a measure of maturation of the immune response(Ma et al., 2012). Each antigen is used at a concentration of 10 μg, asdetermined from published studies and administered with BECC synthesizedmolecules at the same doses used above. Control mice receive antigencoupled with CpG (ODN2095) or antigen with MPL as a positive control(Alving et al., 2012). Overall immune activation is tested by cytokinemultiplex assay on harvested serum as defined in Scheme 2.

In scheme 2 for inoculation and sampling schedule to determineLOS/lipidA adjuvanticity, a baseline serum was obtained at day 0,adjuvant and antigen dose #1 were given at day 14, serum harvestoccurred at day 28, adjuvant and antigen dose #2 and serum harvestoccurred at day 42, serum harvest at day 56, and organ and serum harvestat day 60 (Lung BAL for IgG and IgA Titers and spleen for T cellresponses). To determine if the molecules produce antigen-specificimmunoglobulin(s), serum is analyzed for antigen-specific IgG and IgA byELISA. In addition to total IgG and IgA, one can further explore thesubclasses of IgG antibodies (IgG₁, IgG_(2A), IgG_(2B), IgG_(2C), andIgG₃. These subclasses are influenced by the T helper response, adiversified repertoire of subclasses is indicative of a robust andvaried T helper response. Lung localized antibodies are also analyzed inlavage fluids from mice at day 60. To determine the extent of T cellactivation, spleens and lungs are harvested at day 60 and single cellsuspensions are generated. These preparations are then re-stimulatedwith the administered antigen or the phorbol ester PMA and the ionophoreionomycin as a positive control. These cells are then analyzed bymulticolor flow cytometry for TH1 (IFN-γ, IL-12, TNF-α), TH2 (IL-4,IL-5, IL-13), and TH17 (IL-17, IL-23) recall.

There is assaying of the protective capacity of F1-V antigen coupledwith BECC synthesized molecules. Recent work has shown that couplingF1-V antigen with adjuvants (aluminum hydroxide, the proteasome basedProtillin, and Complete Freund's Adjuvant (CFA)) can induce protectiveimmune responses against a lethal wild type challenge (Heath et al.,1998; Jones et al., 2006; Parent et al., 2005a; Parent et al., 2005b).One can repeat and refine these studies using the novel adjuvants asfollows: C57BL/6 mice are immunized with 10 μg F1-V antigen and ournovel adjuvant as carried out above (Scheme 2). On day 60, mice arechallenged with increasing doses of WT CO92 Yersinia (100 LD₁₀₀, 1,000LD₁₀₀, 10,000 LD₁₀₀) by the IN route. Mice are followed for diseaseprogression and survival for up to 120 days. To confirm theestablishment of long-term protective immunity, adjuvant and antigencombinations that are protective at 60 days are used in a secondarystudy where mice are held for 120 days after this last vaccinationbefore challenge.

There is assaying of the protective capacity of F1-V antigen coupledwith BECC synthesized molecules by the intramuscular route. To determineif the adjuvants can deliver protection when administered by otherroutes one can test vaccination by the intramuscular (IM) route (Joneset al., 2006). huMD2/TLR4 mice are immunized with 10 μg F1-V antigen andthe novel adjuvant by the IM route, as carried out above (Scheme 2). Onday 60, mice are challenged with increasing doses of WT CO92 Yersinia(100 LD₁₀₀, 1,000 LD₁₀₀, 10,000 LD₁₀₀) by the IN route. Mice arefollowed for disease progression and survival for up to 120 days.

Confirmatory studies using humanized huMD2-TLR4 mice are performedMD2/TLR4 complexes from human and mouse have differing reactions tohypoacylated lipid A structures (i.e., murine TLR4 responds to all lipidA structures whereas human TLR4 differentially responds to specificlipid A structures) (Hajjar et al., 2012; Hajjar et al., 2002). Toconfirm that the BECC-synthesized adjuvants can provide protectiveimmune responses when signaling through human MD2/TLR4 complexes,recently developed mice expressing huMD2/TLR4 are used in vaccinationstudies as carried out above in Scheme 2. Briefly, huMD2/TLR4 mice arevaccinated with two doses of 10 μg F1-V antigen along with the noveladjuvant. As a control, mice receive F1-V antigen along with CFA. Serumfrom these mice are analyzed for cytokine production (IL-2, IL-4, IFN-γ,TNF-α, IL-12, IL-8) by multiplex cytokine array. These mice are thenchallenged with increasing doses of WT CO92 Yersinia (100 LD100, 1,000LD100, 10,000 LD100) on day 60 of the experiment. Mice are followed fordisease progression and survival for up to 120 days.

Exemplary Methods with Vertebrate Animals

Female WT C57BL/6 mice (Jackson Laboratories) and huMD2/TLR4 mice(Breeding in house) are used.

Dendritic cell activation studies: Donor mice are harvested for bonemarrow derived dendritic cells. 4 doses of adjuvant (0.5 μg, 5 μg, 50 μgand PBS control)×4 adjuvants (lipid A and LOS from 2 candidatemolecules)×4 mice per experimental group=32 mice. These experiments aredone in triplicate raising the total number of mice to 96.

Adjuvant toxicity studies: 3 doses of adjuvant (0.5 μg, 5 μg, 50 μg)×4adjuvants (lipid A and LOS from 2 candidate molecules)×3 mice pergroup=36 mice+3 PBS dosed control mice=39 mice. These experiments willbe done in triplicate raising the total number of mice to 117.

Antigen specific immune activation studies: 2 antigens (F1-V and PT)×3doses of adjuvant (0.5 μg, 5 μg, 50 μg)×4 adjuvants (lipid A and LOSfrom 2 candidate molecules)×3 mice per group=72 mice+3 PBS dosed+3 CpGdosed control mice+6 CpG and Antigen dosed control mice (3 mice×2antigens) 3 MPL dosed control mice+6 MPL and Antigen dosed control mice(3 mice×2 antigens)=93 mice. These experiments are done in triplicateraising the total number of mice to 289.

Adaptive immune activation and protection studies: Mice are divided into2 test groups (adjuvant alone, adjuvant with F1-V antigen)×4 adjuvants(lipid A and LOS from 2 candidate molecules)×3 mice per group=24 mice.There are 4 control groups for the protection studies (PBS, F1 antigenalone, MPL with F1 antigen, CpG adjuvant with F1 antigen)×3 mice pergroup=12 mice, bringing the total to 36 mice. Mice are challenged by theIN route with 3 escalating doses of fully virulent CO92 strain of Y.pestis (36×3)=108 mice. If these mice demonstrate protection, this studywill also be done in triplicate bringing the number of mice for thisexperiment to 324.

Long term protection studies: Mice are divided into 2 test groups(adjuvant alone, adjuvant with F1-V antigen)×4 adjuvants (lipid A andLOS from 2 candidate molecules)×3 mice per group=24 mice. There is onecontrol group for the protection studies (PBS)=3 mice bringing the totalto 27 mice. Mice are challenged by the IN route with 3 escalating dosesof fully virulent CO92 strain of Y. pestis (27×3)=81 mice. If these micedemonstrate protection, this study will also be done in triplicatebringing the number of mice for this experiment to 243.

Confirmatory studies in huMD2/TLR4 mice: huMD2/TLR4 mice are utilized torule out differential reactions between mouse and human TLR4. Mice aredivided into 2 test groups (adjuvant alone, adjuvant with F1-Vantigen)×4 adjuvants (lipid A and LOS from 2 candidate molecules)×3 miceper group=24 mice. There are 4 control groups for the protection studies(PBS, F1 antigen alone, MPL with F1 antigen, CpG adjuvant with F1antigen)×3 mice per group=12 mice, bringing the total to 36 mice. Miceare challenged by the IN route with 3 escalating doses of fully virulentCO92 strain of Y. pestis (36×3)=108 mice. If these mice demonstrateprotection, this study can be done in triplicate bringing the number ofmice for this experiment to 324.

Protection through IM route vaccine delivery: To determine if IMvaccination of mice can still protect against an IN challenge huMD2/TLR4mice are utilized for a final confirmatory study. Mice are divided into2 test groups (adjuvant alone, adjuvant with F1-V antigen)×4 adjuvants(lipid A and LOS from 2 candidate molecules)×3 mice per group=24 mice.There are 4 control groups for the protection studies (PBS, F1 antigenalone, MPL with F1 antigen, CpG adjuvant with F1 antigen)×3mice pergroup=12 mice, bringing the total to 36 mice. Mice are challenged by theIN route with 3 escalating doses of fully virulent CO92 strain of Y.pestis (36×3)=108 mice. If these mice demonstrate protection, this studywill also be done in triplicate bringing the number of mice for thisexperiment to 324.

Use of the Animals

a. Dendritic Cell Activation:

Donor C57BL/6 mice are used for collection of BMDC. Femurs are harvestedby sterile necropsy and marrow is flushed for in vitro differentiation.

b. Cytotoxicity Studies:

Initially, one can use C57BL/6 mice to determine the cytotoxicity of theadjuvant candidates. Five mice per treatment group are exposed by theintraperitoneal route to increasing doses (0.5 μg, 5 μg, 50 μg) of 1 ofthe 4 adjuvants identified to have the best immunomodulatory potentialin the in vitro testing. All adjuvants are administered in 0.2 ml of PBSas a vehicle. Mice are visually checked twice daily for signs ofdistress or cytotoxicity. Every 14 days peripheral blood is harvestedvia the retro-orbital route for serum immune cytokines and toxicitypanels (ALT, AST, etc). At the end of the test period (60 days), miceare necropsied to collect tissues for microscopic examination of anypossible cytotoxic damage. This analysis is carried out in triplicate.

c. Antigen Specific Immune Activation Studies:

C57BL/6 mice are again used to test the ability of the adjuvantcandidates to stimulate antigen specific immune response. Three mice pertreatment group receive proven non-cytotoxic adjuvants, in the dosestested above, by the intramuscular and intranasal route. As controls 5groups of mice are treated with one of; PBS, MPL, MPL+Antigen, CpG(ODN0295), or CpG+Antigen. Two hours after the injection serum isharvested from the mice and assayed by Luminex and RT-PCR for expressionof pro-inflammatory cytokine and chemokines. This analysis is carriedout in triplicate.

d. Adaptive Immune Activation and Protection Studies:

Adjuvants that have thus far been proven to be both safe andimmunostimulatory are assayed for their ability to stimulate aprotective immune response to a lethal pathogen. Groups of three miceare dosed with either 50 μg of adjuvant or adjuvant as well as 25 μg ofpurified Y. pestis F1-V antigen. Control groups will be dosed with PBS,10 μg F1-V antigen alone, 50 μg MPL with 10 μg F1-V antigen, or 50 μgCpG (ODN0295) with 10 μg F1-V antigen. These mice receive a second“booster” dose 2 weeks after the initial dose. Mice are also assayed fortheir ability to survive a lethal challenge of 100, 1000, and 10000LD_(50's) (LD₅₀˜5 cfu) of the fully virulent CO92 strain of Y. pestis.Four weeks after the boosting inoculation mice are challenged with Y.pestis by the subcutaneous route. Mice are visually checked twice dailyfor signs of disease (eye crusting, piloerection, inactivity,respiratory distress), mice showing disease symptoms are euthanized. Ifmice are able to survive the challenge the studies are repeated intriplicate.

Example 9 Exemplary Lipooligosaccharide/Lipid A-Based Mimetics

FIG. 9 provides a description of individual strains generated, structureof the resultant lipid A, and mass 37° C. Shown therein are examples ofthe Y. pestis KIM6 strains grown at 37° C. (mammalian temperature) withmodified LOS structures. Accompanying major Lipid A structures can beexamined in FIGS. 13-19 generally by referring to the capital letters inthe Structure/Acylation column, e.g. use structure “C” to find thecorresponding major lipid A structure on the following slides showingLipid A structures.

FIG. 10 describes examples of the Y. pestis KIM6 strains grown at 26° C.(flea temperature) with modified LOS structures. Accompanying majorLipid A structures can examined in FIGS. 13-19 generally by referring tothe capital letters in the Structure/Acylation column, e.g. usestructure “C” to find the corresponding major lipid A structure in thefollowing figures showing Lipid A structures.

FIG. 11 shows all results to date from cell stimulations using Y. pestisLOS grown at 37° C. (mammalian temperature) to stimulate the human THP-1Vitamin D3 differentiated alveolar macrophage cell line. The headingsabove the secreted cytokines expressed show the pathway that isactivated after innate immune recognition, e.g. IL-8 signaling isdependent on the MyD88 pathway. These data show that the modified LOScan induce cytokine production through 3 different pathways afterinitial recognition by the Toll-like receptor 4 (TLR4) complex. Thesedata rankings are derived from a stimulus range of 0.1 to 100 ng/ml.

FIG. 12 shows results from cell stimulations using Y. pestis LOS grownat 26° C. (flea temperature) to stimulate the human THP-1 VitaminD3-differentiated alveolar macrophage cell line. The headings above thesecreted cytokines expressed show the pathway that is activated afterinnate immune recognition, e.g., IL-8 signaling is dependent on theMyD88 pathway. These data rankings are derived from a stimulus range of0.1 to 100 ng/ml.

FIGS. 13-20 provide illustrations of exemplary LPS molecules generatedby methods of the disclosure and referred to in FIGS. 9-10. FIG. 21shows control LPS molecules.

REFERENCES

All patents and publications mentioned in the specification areindicative of the level of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

PUBLICATIONS

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

What is claimed is:
 1. A biosynthetic, immunomodulating lipidpolysaccharide compound of formula (I)

wherein R₁ may be H, OH, protonated phosphate, a phosphate salt, a sugarphosphonate, or a mono-, di- or poly-saccharide, R₂ may be H, a sugarphosphonate, or a mono-, di- or poly-saccharide, R₃ may be OH or amono-, di- or poly-saccharide, R₄, R₅, R₆ and R₇ may be an alkyl oralkenyl chain from 7 to 13 carbons, and R₈, R₉, R₁₀ and R₁₁ may be H,OH, or an alkyl or alkenyl ester from 10 to 18 carbons.
 2. The compoundof claim 1, wherein the compound is selected from the group consistingof compounds VIII, IX, X, and XI


3. A kit, comprising one or more of the compounds of claim
 1. 4. The kitof claim 3, further comprising one or both of an antigen and apharmaceutically acceptable carrier.
 5. A biosynthetic, immunomodulatinglipid polysaccharide compound of the formula (XIV):